Figure 3 ( A ) Immunofluorescence staining of FTCD and GM130 , a Golgi marker . HepG2 cells were fixed , permeabilized , stained with a monoclonal antibody to FTCD , a polyclonal antibody to GM130 and DAPI , and observed by confocal microscopy . Scale bar = 10 μm . ( B ) Depletion of FTCD by FTCD siRNA duplexes . HepG2 cells were either mock transfected with water or transfected with two distinct siRNA duplexes specific to FTCD . After incubation for 48 hrs , the cells were analyzed by Western blotting with antibodies to FTCD and α - tubulin . ( C ) The Golgi in cells which were depleted of either FTCD or p47 . HepG2 cells , which were treated with either FTCD siRNAs , p47 siRNA or mock for 48 hrs , were fixed and stained with a monoclonal antibody to GM130 . Scale bar = 10 μm . ( D ) The results of quantification of ( C ) . The values are from five sets of independent experiments . Results are expressed as the mean ± SD ( n = 5 ) , with 100 cells counted in each group of each set . Asterisks indicate a significant difference at P < 0 . 01 compared with the mock - treated cells ( Bonferroni method ) . ( E ) Representative EM images of the Golgi in HepG2 cells treated with either mock or FTCD siRNAs for 42 hrs . Scale bar = 0 . 5 μm . ( F ) The results of quantification of ( E ) . Golgi membranes in HepG2 cells were classified into cisternae , vesicles and tubules , and counted as previously described ( Shorter & Warren , 1999 ) . The results are shown as the mean ± SD of five sets of independent experiments , with 15 - 20 cells counted in each group in each independent experiment . An asterisk indicates a significant difference ( P < 0 . 05 ) compared with the mock - treated cells in each category ( Bonferroni method ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Immunofluorescence staining of FTCD and GM130, a Golgi marker. HepG2 cells were fixed, permeabilized, stained with a monoclonal antibody to FTCD, a polyclonal antibody to GM130 and DAPI, and observed by confocal microscopy. Scale bar = 10 μm.(B) Depletion of FTCD by FTCD siRNA duplexes. HepG2 cells were either mock transfected with water or transfected with two distinct siRNA duplexes specific to FTCD. After incubation for 48 hrs, the cells were analyzed by Western blotting with antibodies to FTCD and α-tubulin.(C) The Golgi in cells which were depleted of either FTCD or p47. HepG2 cells, which were treated with either FTCD siRNAs, p47 siRNA or mock for 48 hrs, were fixed and stained with a monoclonal antibody to GM130. Scale bar = 10 μm. (D) The results of quantification of (C). The values are from five sets of independent experiments. Results are expressed as the mean±SD (n=5), with 100 cells counted in each group of each set. Asterisks indicate a significant difference at P<0.01 compared with the mock-treated cells (Bonferroni method). (E) Representative EM images of the Golgi in HepG2 cells treated with either mock or FTCD siRNAs for 42 hrs. Scale bar = 0.5 μm. (F) The results of quantification of (E). Golgi membranes in HepG2 cells were classified into cisternae, vesicles and tubules, and counted as previously described (Shorter & Warren, 1999). The results are shown as the mean±SD of five sets of independent experiments, with 15-20 cells counted in each group in each independent experiment. An asterisk indicates a significant difference (P<0.05) compared with the mock-treated cells in each category (Bonferroni method).


Figure 4 ( A ) The localization of FTCD in mitotic cells . Mitotic HepG2 cells were collected by flushing and cultured on coated coverslips for 10 - 40 mins . The cells were fixed , stained with a monoclonal antibody to FTCD and polyclonal antibodies to GM130 and α - tubulin , and observed by confocal microscopy . Panels d , h , and l indicate the images of α - tubulin and the other panels show the distribution of FTCD or / and GM130 . Scale bar = 10 μm . Arrowheads indicate the colocalization of FTCD and GM130 . ( B ) The effects of FTCD siRNA treatment on Golgi reassembly at the end of mitosis . HepG2 cells were treated with either FTCD siRNA ( FTCD siRNA 2 duplex ) or mock for 48 hrs before the collection of mitotic cells . The collected mitotic cells were cultured for 35 mins and then fixed . The cells were stained with a polyclonal antibody to GM130 and a monoclonal antibody to α - tubulin , and observed by confocal microscopy . Panels a - p display representative images . Scale bar = 10 μm . ( C ) Rescue experiments in FTCD siRNA - treated HepG2 cells . The indicated HA - tagged FTCDs , which were insensitive to FTCD siRNA , were expressed 25 hrs after the treatment of cells with FTCD siRNA2 . The cells were further cultured for 23 hrs and then used for the collection of mitotic cells . The collected mitotic cells were cultured for 35 mins and fixed . The cells were stained with a monoclonal antibody to α - tubulin ( panels g and h ) and polyclonal antibodies to GM130 and HA ( panels a - f ) . Scale bar = 10 μm . ( D ) The cells at cytokinesis in ( B ) and ( C ) were analyzed . The results are shown as the mean ± SD of five sets of independent experiments , with 100 cells counted in each group in each independent experiment . Asterisks indicate a significant difference ( P < 0 . 01 ) compared with the mock - treated cells ( Bonferroni method ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) The localization of FTCD in mitotic cells. Mitotic HepG2 cells were collected by flushing and cultured on coated coverslips for 10-40 mins. The cells were fixed, stained with a monoclonal antibody to FTCD and polyclonal antibodies to GM130 and α-tubulin, and observed by confocal microscopy. Panels d, h, and l indicate the images of α-tubulin and the other panels show the distribution of FTCD or/and GM130. Scale bar = 10 μm. Arrowheads indicate the colocalization of FTCD and GM130.(B) The effects of FTCD siRNA treatment on Golgi reassembly at the end of mitosis. HepG2 cells were treated with either FTCD siRNA (FTCD siRNA 2 duplex) or mock for 48 hrs before the collection of mitotic cells. The collected mitotic cells were cultured for 35 mins and then fixed. The cells were stained with a polyclonal antibody to GM130 and a monoclonal antibody to α-tubulin, and observed by confocal microscopy. Panels a-p display representative images. Scale bar = 10 μm.(C) Rescue experiments in FTCD siRNA-treated HepG2 cells. The indicated HA-tagged FTCDs, which were insensitive to FTCD siRNA, were expressed 25 hrs after the treatment of cells with FTCD siRNA2. The cells were further cultured for 23 hrs and then used for the collection of mitotic cells. The collected mitotic cells were cultured for 35 mins and fixed. The cells were stained with a monoclonal antibody to α-tubulin (panels g and h) and polyclonal antibodies to GM130 and HA (panels a-f). Scale bar = 10 μm.(D) The cells at cytokinesis in (B) and (C) were analyzed. The results are shown as the mean±SD of five sets of independent experiments, with 100 cells counted in each group in each independent experiment. Asterisks indicate a significant difference (P<0.01) compared with the mock-treated cells (Bonferroni method).


Figure 5 ( A ) p47 requires its association with FTCD for its localization to the Golgi at the end of mitosis . HepG2 cells were treated with FTCD siRNA and then the indicated HA - tagged FTCDs were expressed as described in Figure 4C . The mitotic cells were collected , cultured for 35 mins , and then fixed . The cells were stained with polyclonal antibodies to p47 and HA and a monoclonal antibody to GM130 , and observed by confocal microscopy . Panels a - l show the distribution of p47 or / and GM130 and panels m - o show HA - FTCD expression . Scale bar = 10 μm . ( B ) Insets in ( A ) . p47 ( red ) and the GM130 ( a Golgi marker , green ) were visualized . Scale bar = 5 μm . ( C ) The results of quantification of ( A ) . The results are shown as the mean ± SD of five sets of independent experiments , with 100 cells counted in each group in each independent experiments . Asterisks indicate a significant difference at P < 0 . 01 compared with the mock - treated cells ( Bonferroni method ) . ( D ) p97 requires its association with FTCD for its localization to the Golgi at the end of mitosis . The cells were treated in the same way as ( A ) and stained with polyclonal antibodies to p97 and HA and a monoclonal antibody to GM130 . Panels a - l show the distribution of p97 or / and GM130 and panels m - o show HA - FTCD expression . Scale bar = 10 μm . ( E ) Insets in ( D ) . p97 ( red ) and the GM130 ( a Golgi marker , green ) were visualized . Scale bar = 5 μm . ( F ) The results of quantification of ( D ) . The results are shown as the mean ± SD of five sets of independent experiments , with 100 cells counted in each group in each independent experiments . Asterisks indicate a significant difference at P < 0 . 01 compared with the mock - treated cells ( Bonferroni method ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) p47 requires its association with FTCD for its localization to the Golgi at the end of mitosis. HepG2 cells were treated with FTCD siRNA and then the indicated HA-tagged FTCDs were expressed as described in Figure 4C. The mitotic cells were collected, cultured for 35 mins, and then fixed. The cells were stained with polyclonal antibodies to p47 and HA and a monoclonal antibody to GM130, and observed by confocal microscopy. Panels a-l show the distribution of p47 or/and GM130 and panels m-o show HA-FTCD expression. Scale bar = 10 μm. (B) Insets in (A). p47 (red) and the GM130 (a Golgi marker, green) were visualized. Scale bar = 5 μm. (C) The results of quantification of (A). The results are shown as the mean±SD of five sets of independent experiments, with 100 cells counted in each group in each independent experiments. Asterisks indicate a significant difference at P<0.01 compared with the mock-treated cells (Bonferroni method). (D) p97 requires its association with FTCD for its localization to the Golgi at the end of mitosis. The cells were treated in the same way as (A) and stained with polyclonal antibodies to p97 and HA and a monoclonal antibody to GM130. Panels a-l show the distribution of p97 or/and GM130 and panels m-o show HA-FTCD expression. Scale bar = 10 μm. (E) Insets in (D). p97 (red) and the GM130 (a Golgi marker, green) were visualized. Scale bar = 5 μm. (F) The results of quantification of (D). The results are shown as the mean±SD of five sets of independent experiments, with 100 cells counted in each group in each independent experiments. Asterisks indicate a significant difference at P<0.01 compared with the mock-treated cells (Bonferroni method).


Figure 6 ( A ) Anti - FTCD antibodies inhibit p97 / p47 - mediated Golgi membrane fusion . Golgi fragments were mixed with the indicated components ( p97 , 60 μg / ml ; p47 , 30 μg / ml ; p37 , 24 μg / ml ; p115 , 40 μg / ml ) together with either anti - FTCD or anti - p115 antibodies . Results are expressed as mean + SD of five sets of independent experiments ; 0 % and 100 % represent the buffer only ( 25 . 9 % in cisternal membranes ) and p97 / p47 with no antibodies ( ' + none ' , 48 . 8 % ) , respectively . Asterisks indicate a significant difference at P < 0 . 01 compared with the no antibody groups ( ' + none ' ) ( Bonferroni method ) . ( B ) p47 ( E86K , E88K ) , which lacks binding affinity to FTCD , does not function in p97 / p47 - mediated Golgi membrane fusion . Golgi fragments were incubated with p97 ( 60 μg / ml ) and p47wt / mutant ( 30 μg / ml ) . Data are shown as the mean + SD of five sets of independent experiments ; 0 % and 100 % represent the buffer only ( 27 . 3 % in cisternal membranes ) and p97 + p47wt ( ' + p47wt ' , 50 . 9 % ) , respectively . Asterisks indicate a significant difference at P < 0 . 01 compared with the group in which p47wt as well as p97 were added ( ' + p47wt ' ) ( Bonferroni method ) . ( C ) p97 ( V722K , E724K ) , which lacks binding affinity to FTCD , does not function in p97 / p47 - mediated Golgi membrane fusion . Golgi fragments were incubated with the following components : p97wt / mutant , 60 μg / ml ; p47 , 30 μg / ml ; p37 , 24 μg / ml ; p115 , 40 μg / ml . Data are shown as the mean + SD of five sets of independent experiments ; 0 % and 100 % represent the buffer only ( 29 . 5 % in cisternal membranes ) , and p47 + p97wt ( ' + p97wt ' . 57 . 5 % ) , respectively . An asterisk indicates a significant difference at P < 0 . 01 compared with the group in which p97wt as well as p47 were added ( ' + p97wt ' ) ( Bonferroni method ) . ( D ) Panels a - e show representative EM images . Scale bar = 0 . 5 μm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O

Figure 6(A) Anti-FTCD antibodies inhibit p97/p47-mediated Golgi membrane fusion. Golgi fragments were mixed with the indicated components (p97, 60 μg/ml; p47, 30 μg/ml; p37, 24 μg/ml; p115, 40 μg/ml) together with either anti-FTCD or anti-p115 antibodies. Results are expressed as mean+SD of five sets of independent experiments; 0% and 100% represent the buffer only (25.9% in cisternal membranes) and p97/p47 with no antibodies ('+ none', 48.8%), respectively. Asterisks indicate a significant difference at P<0.01 compared with the no antibody groups ('+none') (Bonferroni method).(B) p47(E86K, E88K), which lacks binding affinity to FTCD, does not function in p97/p47-mediated Golgi membrane fusion. Golgi fragments were incubated with p97 (60 μg/ml) and p47wt/mutant (30 μg/ml). Data are shown as the mean+SD of five sets of independent experiments; 0% and 100% represent the buffer only (27.3% in cisternal membranes) and p97+p47wt ('+p47wt', 50.9%), respectively. Asterisks indicate a significant difference at P<0.01 compared with the group in which p47wt as well as p97 were added ('+p47wt') (Bonferroni method).(C) p97(V722K,E724K), which lacks binding affinity to FTCD, does not function in p97/p47-mediated Golgi membrane fusion. Golgi fragments were incubated with the following components: p97wt/mutant, 60 μg/ml; p47, 30 μg/ml; p37, 24 μg/ml; p115, 40 μg/ml. Data are shown as the mean+SD of five sets of independent experiments; 0% and 100% represent the buffer only (29.5% in cisternal membranes), and p47+p97wt ('+p97wt'. 57.5%), respectively. An asterisk indicates a significant difference at P<0.01 compared with the group in which p97wt as well as p47 were added ('+p97wt') (Bonferroni method).(D) Panels a-e show representative EM images. Scale bar = 0.5 μm.


Figure 7 ( A ) Two sets of FTCD molecules and a p97 / p47 complex form a quaternary complex . His - tagged FTCD was biotinylated and immobilized on streptavidin beads ( His - FTCD - beads ) . The beads ( 1 . 0 μg ) were incubated with Flag - tagged FTCD ( 2 . 0 μg ) in the absence or presence of p47 ( 1 . 0 μg ) and p97 ( 2 . 0 μg ) . The blots were probed with HRP - conjugated avidin and antibodies to Flag - tag , p97 and p47 . ( B ) Formation of the quaternary complex in the presence of nucleotides . His - FTCD - beads ( 1 . 0 μg ) were incubated with Flag - tagged FTCD ( 2 . 0 μg ) , p97 ( 2 . 0 μg ) and p47 ( 1 . 0 μg ) in the presence of nucleotides ( 1 mM ) . The blots were probed as described in ( A ) . ( C ) Mutants that lack binding affinity disrupt the formation of the quaternary complex . His - FTCD - beads ( 1 . 0 μg ) and Flag - tagged FTCD ( 2 . 0 μg ) were incubated in the presence of AMP - PNP together with p97wt / mutant ( 2 . 0 μg ) and p47wt / mutant ( 1 . 0 μg ) . The blots were probed as described in ( A ) . ( D ) Negative stain electron microscopy of the quaternary complex . The panels show class averages of centered and aligned negative stain particles for the different populations present within the dataset , i . e . , the full quaternary complex FTCD - p97 / p47 - FTCD ( left panel ) , and its partial complexes ( two variations , middle two panels ) . Class averages contain approximately 20 images per class . A class average showing a p97 / p47 side view from cryo - EM data and a projection generated from FTCD structure ( PDB code 1TT9 ) have been included for comparisons . Brackets and arrow heads indicate the p97 hexamer and the FTCD octamer , respectively . Scale bar = 15 nm . ( E ) FTCDwt - beads are aggregated in the presence of both p97 and p47 . Biotinylated FTCDwt and FTCD ( R382A ) were immobilized on streptavidin magnetic beads to form FTCDwt - beads and FTCD ( R382A ) - beads , respectively . The beads were incubated with the indicated proteins ( p97 , 0 . 1 μg / μl ; p47 , 0 . 1 μg / μl ; p37 , 0 . 1 μg / μl ) in the presence of AMP - PNP ( 1 mM ) , and observed under a bright - field microscope . Scale bar = 50 μm . ( F ) The results of quantification of ( E ) . The percentages of beads in the aggregates were calculated . Results are expressed as the mean + SD of ten sets of independent experiments . An asterisk indicates a significant difference at P < 0 . 01 compared with the others ( Bonferroni method ) . ( G ) p97 / p47 - mediated aggregation of FTCDwt - beads is nucleotide - dependent . FTCDwt - beads were incubated together with p97 ( 0 . 1 μg / μl ) and p47 ( 0 . 1 μg / μl ) in the presence of the indicated nucleotides ( 1mM ) . The representative images are presented in Figure EV1E . Data are shown as the mean + SD of nine sets of independent experiments . An asterisk and hash tags indicate significant differences at P < 0 . 01 compared with the others and the negative control ( ' none ' ) , respectively ( Bonferroni method ) . ( H ) p47 and p97 mutants that lack binding affinity inhibit p97 / p47 - mediated aggregation of FTCDwt - beads . FTCDwt - beads were incubated with p97wt / mutant ( 0 . 1 μg / μl ) and p47wt / mutants ( 0 . 1 μg / μl ) in the presence of AMP - PNP . The representative images are presented in Figure EV1F . Data are shown as the mean + SD of nine sets of independent experiments . Asterisks indicate a significant difference at P < 0 . 01 compared with the positive control ( ' p97wt + p47wt ' ) ( Bonferroni method ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A) Two sets of FTCD molecules and a p97/p47 complex form a quaternary complex. His-tagged FTCD was biotinylated and immobilized on streptavidin beads (His-FTCD-beads). The beads (1.0 μg) were incubated with Flag-tagged FTCD (2.0 μg) in the absence or presence of p47 (1.0 μg) and p97 (2.0 μg). The blots were probed with HRP-conjugated avidin and antibodies to Flag-tag, p97 and p47.(B) Formation of the quaternary complex in the presence of nucleotides. His-FTCD-beads (1.0 μg) were incubated with Flag-tagged FTCD (2.0 μg), p97 (2.0 μg) and p47 (1.0 μg) in the presence of nucleotides (1 mM). The blots were probed as described in (A).(C) Mutants that lack binding affinity disrupt the formation of the quaternary complex. His-FTCD-beads (1.0 μg) and Flag-tagged FTCD (2.0 μg) were incubated in the presence of AMP-PNP together with p97wt/mutant (2.0 μg) and p47wt/mutant (1.0 μg). The blots were probed as described in (A).(D) Negative stain electron microscopy of the quaternary complex. The panels show class averages of centered and aligned negative stain particles for the different populations present within the dataset, i.e., the full quaternary complex FTCD-p97/p47-FTCD (left panel), and its partial complexes (two variations, middle two panels). Class averages contain approximately 20 images per class. A class average showing a p97/p47 side view from cryo-EM data and a projection generated from FTCD structure (PDB code 1TT9) have been included for comparisons. Brackets and arrow heads indicate the p97 hexamer and the FTCD octamer, respectively. Scale bar = 15 nm.(E) FTCDwt-beads are aggregated in the presence of both p97 and p47. Biotinylated FTCDwt and FTCD(R382A) were immobilized on streptavidin magnetic beads to form FTCDwt-beads and FTCD(R382A)-beads, respectively. The beads were incubated with the indicated proteins (p97, 0.1 μg/μl; p47, 0.1 μg/μl; p37, 0.1 μg/μl) in the presence of AMP-PNP (1 mM), and observed under a bright-field microscope. Scale bar = 50 μm. (F) The results of quantification of (E). The percentages of beads in the aggregates were calculated. Results are expressed as the mean+SD of ten sets of independent experiments. An asterisk indicates a significant difference at P<0.01 compared with the others (Bonferroni method). (G) p97/p47-mediated aggregation of FTCDwt-beads is nucleotide-dependent. FTCDwt-beads were incubated together with p97 (0.1 μg/μl) and p47 (0.1 μg/μl) in the presence of the indicated nucleotides (1mM). The representative images are presented in Figure EV1E. Data are shown as the mean+SD of nine sets of independent experiments. An asterisk and hash tags indicate significant differences at P<0.01 compared with the others and the negative control ('none'), respectively (Bonferroni method).(H) p47 and p97 mutants that lack binding affinity inhibit p97/p47-mediated aggregation of FTCDwt-beads. FTCDwt-beads were incubated with p97wt/mutant (0.1 μg/μl) and p47wt/mutants (0.1 μg/μl) in the presence of AMP-PNP. The representative images are presented in Figure EV1F. Data are shown as the mean+SD of nine sets of independent experiments. Asterisks indicate a significant difference at P<0.01 compared with the positive control ('p97wt+p47wt') (Bonferroni method).


Figure 8 ( A ) The expression of FTCDwt - HA - MAO causes the aggregation of mitochondria . HeLa Tet - off cells inducibly expressing either FTCDwt - HA - MAO or FTCD ( R382A ) - HA - MAO were cultured in DOX - free medium for 48 hrs . Cells which were cultured in the DOX - containing medium were used as a control . The cells were fixed and stained with a monoclonal antibody to mitochondria and a polyclonal antibody to HA , followed by observation by confocal microscopy . Panels a - j display representative images . Scale bar = 10 μm . ( B ) The results of quantification of ( A ) . Results are shown as the mean ± SD of five sets of independent experiments , with 100 cells counted in each group in each independent experiment . An asterisk indicates a significant difference at P < 0 . 01 compared with the others ( Bonferroni method ) . ( C ) Representative EM images of a cell expressing FTCDwt - HA - MAO . The HeLa Tet - off cells inducibly expressing FTCDwt - HA - MAO were cultured for 48 hrs in the absence of DOX , and used for EM observation . Panel b displays the inset in panel a . Arrowheads show narrow gaps between mitochondria . Scale bar = 0 . 2 μm . The low magnification image is presented in Figure EV2C . ( D ) Endogenous p47 and p97 are necessary for mitochondria aggregation mediated by FTCDwt - HA - MAO . HeLa Tet - off cells inducibly expressing FTCDwt - HA - MAO were transfected with either mock , p47 siRNA or p97 siRNA duplexes and cultured for 24 hrs . The cells were further cultured in DOX - free medium for 48 hrs for the induction of FTCDwt - HA - MAO , and then analyzed as in ( A ) . Panels a - l display representative images . Scale bar = 10 μm . ( E ) The results of quantification of ( D ) . Results are shown as the mean ± SD of five sets of independent experiments , with 100 cells counted in each group in each independent experiment . An asterisk indicates a significant difference at P < 0 . 01 compared with the others ( Bonferroni method ) . ( F ) Binding between FTCD and p47 and between p47 and p97 is necessary for mitochondria aggregation mediated by FTCDwt - HA - MAO . The HeLa Tet - off cells inducibly expressing FTCDwt - HA - MAO were transfected with mammalian expression constructs of siRNA - insensitive Flag - tagged p47wt / mutants at the same time as the treatment of p47 siRNA , and cultured for 24 hrs . The cells were further cultured in DOX - free medium for 48 hrs for the induction of FTCD - HA - MAO . After fixation , the cells were visualized with a monoclonal antibody to mitochondria and polyclonal antibodies to HA and Flag . Panels a - l display representative images . Scale bar = 10 μm . ( G ) Binding between FTCD and p97 is necessary for mitochondria aggregation mediated by FTCDwt - HA - MAO . The HeLa Tet - off cells inducibly expressing FTCDwt - HA - MAO were transfected with the mammalian expression construct of siRNA - insensitive Flag - tagged p97wt / mutant at the same time as treatment with p97 siRNA . The following procedures were the same as in ( F ) . Panels a - i display representative images . Scale bar = 10 μm . ( H ) The results of quantification of ( F ) and ( G ) . Results are shown as the mean ± SD of five sets of independent experiments , with 100 cells counted in each group in each independent experiment . Asterisks indicate a significant difference at P < 0 . 01 compared with siRNA treatment alone ( ' none ' ) and compared with mutant expression ( Bonferroni method ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8(A) The expression of FTCDwt-HA-MAO causes the aggregation of mitochondria. HeLa Tet-off cells inducibly expressing either FTCDwt-HA-MAO or FTCD(R382A)-HA-MAO were cultured in DOX-free medium for 48 hrs. Cells which were cultured in the DOX-containing medium were used as a control. The cells were fixed and stained with a monoclonal antibody to mitochondria and a polyclonal antibody to HA, followed by observation by confocal microscopy. Panels a-j display representative images. Scale bar = 10 μm. (B) The results of quantification of (A). Results are shown as the mean±SD of five sets of independent experiments, with 100 cells counted in each group in each independent experiment. An asterisk indicates a significant difference at P<0.01 compared with the others (Bonferroni method). (C) Representative EM images of a cell expressing FTCDwt-HA-MAO. The HeLa Tet-off cells inducibly expressing FTCDwt-HA-MAO were cultured for 48 hrs in the absence of DOX, and used for EM observation. Panel b displays the inset in panel a. Arrowheads show narrow gaps between mitochondria. Scale bar = 0.2 μm. The low magnification image is presented in Figure EV2C.(D) Endogenous p47 and p97 are necessary for mitochondria aggregation mediated by FTCDwt-HA-MAO. HeLa Tet-off cells inducibly expressing FTCDwt-HA-MAO were transfected with either mock, p47 siRNA or p97 siRNA duplexes and cultured for 24 hrs. The cells were further cultured in DOX-free medium for 48 hrs for the induction of FTCDwt-HA-MAO, and then analyzed as in (A). Panels a-l display representative images. Scale bar = 10 μm. (E) The results of quantification of (D). Results are shown as the mean±SD of five sets of independent experiments, with 100 cells counted in each group in each independent experiment. An asterisk indicates a significant difference at P<0.01 compared with the others (Bonferroni method). (F) Binding between FTCD and p47 and between p47 and p97 is necessary for mitochondria aggregation mediated by FTCDwt-HA-MAO. The HeLa Tet-off cells inducibly expressing FTCDwt-HA-MAO were transfected with mammalian expression constructs of siRNA-insensitive Flag-tagged p47wt/mutants at the same time as the treatment of p47 siRNA, and cultured for 24 hrs. The cells were further cultured in DOX-free medium for 48 hrs for the induction of FTCD-HA-MAO. After fixation, the cells were visualized with a monoclonal antibody to mitochondria and polyclonal antibodies to HA and Flag. Panels a-l display representative images. Scale bar = 10 μm. (G) Binding between FTCD and p97 is necessary for mitochondria aggregation mediated by FTCDwt-HA-MAO. The HeLa Tet-off cells inducibly expressing FTCDwt-HA-MAO were transfected with the mammalian expression construct of siRNA-insensitive Flag-tagged p97wt/mutant at the same time as treatment with p97 siRNA. The following procedures were the same as in (F). Panels a-i display representative images. Scale bar = 10 μm. (H) The results of quantification of (F) and (G). Results are shown as the mean±SD of five sets of independent experiments, with 100 cells counted in each group in each independent experiment. Asterisks indicate a significant difference at P<0.01 compared with siRNA treatment alone ('none') and compared with mutant expression (Bonferroni method).


Figure 9 ( A ) FTCD located in mitochondria causes mitochondria aggregation during mitosis . Mitotic cells were collected by flushing from HeLa Tet - off cells in which either FTCDwt - HA - MAO or FTCD ( R382A ) - HA - MAO had been induced for 24 hrs . The mitotic cells were further cultured for 4 hrs to enable complete cytokinesis . Cells in which FTCDwt - HA - MAO was induced for 28 hrs without synchronization were used as the asynchronous control group . Cells were fixed and visualized with a monoclonal antibody to mitochondria and a polyclonal antibody to HA . Panels a - f display representative images . Scale bar = 10 μm . ( B ) The results of quantification of ( A ) . Results are shown as the mean ± SD of seven sets of independent experiments , with 100 cells counted in each group in each independent experiment . An asterisk indicates a significant difference at P < 0 . 01 compared with the others ( Bonferroni method ) . ( C ) A single living cell expressing FTCDwt - HA - MAO was tracked during mitosis . Mitotic cells were collected by flushing from the HeLa Tet - off cells inducibly expressing FTCDwt - HA - MAO , and cultured in the absence or presence of DOX . The cells were stained with MitoTracker at the 22 hr time point and confocal images of 30 - 40 cells were randomly taken at the 23 hr time point . From the 23 hr to 27 hr time point , cells were tracked in the bright field every 30 mins . At the 27 hr time point , confocal images were taken of the 21 - 38 cells that were successfully tracked . Panels a - j display representative images . Asterisks indicate the tracked cells . Scale bar = 10 μm . ( D ) The results of quantification of ( C ) . The experiment was repeated five times and each line shows the average percentage of an independent experiment . The percentages of cells that underwent mitosis are as follows : 41 . 5 % in the DOX ( - ) group and 54 . 7 % in the DOX ( + ) group . ( E ) FTCD located in mitochondria causes mitochondria aggregation at the end of mitosis . Mitotic cells were collected from the HeLa Tet - off cells inducibly expressing FTCDwt - HA - MAO and cultured in the absence or presence of DOX as in ( C ) . Cells were stained with MitoTracker at the 23hr time point and observed by confocal microscopy from the 24 hr to 26 hr time point . Panels a - t display representative images . Scale bar = 10 μm . ( F ) The results of quantification of ( E ) . Cells were classified by their cell cycle phases into the following three categories : prometaphase / metaphase , anaphase / telophase and cytokinesis . Results are shown as the mean ± SD of three sets of independent experiments , with 10 - 14 cells counted in each group in each independent experiment . Asterisks indicate a significant difference at P < 0 . 01 compared with the DOX ( + ) group and with the prometaphase / metaphase category ( Bonferroni method ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 9(A) FTCD located in mitochondria causes mitochondria aggregation during mitosis. Mitotic cells were collected by flushing from HeLa Tet-off cells in which either FTCDwt-HA-MAO or FTCD(R382A)-HA-MAO had been induced for 24 hrs. The mitotic cells were further cultured for 4 hrs to enable complete cytokinesis. Cells in which FTCDwt-HA-MAO was induced for 28 hrs without synchronization were used as the asynchronous control group. Cells were fixed and visualized with a monoclonal antibody to mitochondria and a polyclonal antibody to HA. Panels a-f display representative images. Scale bar = 10 μm. (B) The results of quantification of (A). Results are shown as the mean±SD of seven sets of independent experiments, with 100 cells counted in each group in each independent experiment. An asterisk indicates a significant difference at P<0.01 compared with the others (Bonferroni method). (C) A single living cell expressing FTCDwt-HA-MAO was tracked during mitosis. Mitotic cells were collected by flushing from the HeLa Tet-off cells inducibly expressing FTCDwt-HA-MAO, and cultured in the absence or presence of DOX. The cells were stained with MitoTracker at the 22 hr time point and confocal images of 30-40 cells were randomly taken at the 23 hr time point. From the 23 hr to 27 hr time point, cells were tracked in the bright field every 30 mins. At the 27 hr time point, confocal images were taken of the 21-38 cells that were successfully tracked. Panels a-j display representative images. Asterisks indicate the tracked cells. Scale bar = 10 μm. (D) The results of quantification of (C). The experiment was repeated five times and each line shows the average percentage of an independent experiment. The percentages of cells that underwent mitosis are as follows: 41.5 % in the DOX(-) group and 54.7 % in the DOX(+) group. (E) FTCD located in mitochondria causes mitochondria aggregation at the end of mitosis. Mitotic cells were collected from the HeLa Tet-off cells inducibly expressing FTCDwt-HA-MAO and cultured in the absence or presence of DOX as in (C). Cells were stained with MitoTracker at the 23hr time point and observed by confocal microscopy from the 24 hr to 26 hr time point. Panels a-t display representative images. Scale bar = 10 μm.(F) The results of quantification of (E). Cells were classified by their cell cycle phases into the following three categories: prometaphase/metaphase, anaphase/telophase and cytokinesis. Results are shown as the mean±SD of three sets of independent experiments, with 10-14 cells counted in each group in each independent experiment. Asterisks indicate a significant difference at P<0.01 compared with the DOX(+) group and with the prometaphase/metaphase category (Bonferroni method).


Figure 2A , Heat map of all reported cancer blood metabolites ( each dark blue mark denotes that the metabolite was reported to be increased or decreased ) in various cancer types , illustrating that most individual studies report only a small subset of all previously measured metabolites . It is typically not described whether the metabolites that were not reported ( white " empty " space ) were not measured , or measured but not reported . Labelings : X - axis ( top ) : ribbon colour code , denoting the cancer type ( right ; indicated by red arrow ) ; X - axis ( bottom ) : cohorts , arranged from 1 to 71 ; Y - axis ( right ) : all 1 , 206 metabolites reported in at least one of the studies ; Y - axis ( left ) : ribbon colour code , denoting the metabolite class ( amino acids , carbohydrates , etc ; " other " refers to all other metabolites than the listed classes ) . B , Vote counting of cancer blood metabolites ( reported in at least six cohorts ) showed consistently deregulated metabolites . Blue bars : decreased metabolites ; red bars : increased metabolites . * ( asterisk ) in front of the name of the metabolite indicates a statistical trend ( p < 0 . 1 ) ; red arrowheads denote metabolites mentioned in the main text . C , D , Volcano plot of bloodmetabolites ( C ) and tissuemetabolites ( D ) , reported in at least six cancer studies , with the vote counting score on the X - axis and the - log10 adjusted p - value on the Y - axis . Cyan indicates deregulated metabolites that show a trend ( p < 0 . 1 ; for blood , corresponding to metabolites marked with * in panel 2B ; for tissue , corresponding to metabolites marked with * in Figure 3 ) or statistical significance ( p < 0 . 05 ; above black dashed horizontal line ) ; red indicates metabolites with a p - value > 0 . 1 . A subset of metabolites is annotated ( see Table EV4 ( blood ) and EV5 ( tissue ) for full annotation ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A, Heat map of all reported cancer blood metabolites (each dark blue mark denotes that the metabolite was reported to be increased or decreased) in various cancer types, illustrating that most individual studies report only a small subset of all previously measured metabolites. It is typically not described whether the metabolites that were not reported (white "empty" space) were not measured, or measured but not reported. Labelings: X-axis (top): ribbon colour code, denoting the cancer type (right; indicated by red arrow); X-axis (bottom): cohorts, arranged from 1 to 71; Y-axis (right): all 1,206 metabolites reported in at least one of the studies; Y-axis (left): ribbon colour code, denoting the metabolite class (amino acids, carbohydrates, etc; "other" refers to all other metabolites than the listed classes).B, Vote counting of cancer blood metabolites (reported in at least six cohorts) showed consistently deregulated metabolites. Blue bars: decreased metabolites; red bars: increased metabolites. * (asterisk) in front of the name of the metabolite indicates a statistical trend (p < 0.1); red arrowheads denote metabolites mentioned in the main text.C,D, Volcano plot of bloodmetabolites (C) and tissuemetabolites (D), reported in at least six cancer studies, with the vote counting score on the X-axis and the -log10 adjusted p-value on the Y-axis. Cyan indicates deregulated metabolites that show a trend (p < 0.1; for blood, corresponding to metabolites marked with * in panel 2B; for tissue, corresponding to metabolites marked with * in Figure 3) or statistical significance (p < 0.05; above black dashed horizontal line); red indicates metabolites with a p-value > 0.1. A subset of metabolites is annotated (see Table EV4 (blood) and EV5 (tissue) for full annotation).


Figure 3Vote counting of cancer tissue metabolites ( reported in at least six cohorts ) showed consistently deregulated metabolites . Blue bars : decreased metabolites ; red bars : increased metabolites . * ( asterisk ) in front of the name of the metabolite indicates a statistical trend ( p < 0 . 1 ) ; red arrows denote key metabolites mentioned in the main text ( see Table EV5 for full annotation ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Vote counting of cancer tissue metabolites (reported in at least six cohorts) showed consistently deregulated metabolites. Blue bars: decreased metabolites; red bars: increased metabolites. * (asterisk) in front of the name of the metabolite indicates a statistical trend (p < 0.1); red arrows denote key metabolites mentioned in the main text (see Table EV5 for full annotation).


Figure 1Representative images ( top ) and quantification ( bottom ) of TRF1 nuclear fluorescence of H676 cells treated with the indicated compounds for 24h at 1 μM . Scale bars , 5μm . Data are representative of n = 3 biological replicates . ( C ) Western blot images ( top ) and TRF1 protein levels ( bottom ) of h676 cells treated with the indicated compounds for 24h at 1 μM . Data are representative of n = 3 ( PLKi , HSP90i and RTKi ) and n = 4 ( AuroraI , mTOR , CDKi , Docetaxel , Gemcitabine , ERKi , MEKi ) biological replicates .

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Figure 1Representative images (top) and quantification (bottom) of TRF1 nuclear fluorescence of H676 cells treated with the indicated compounds for 24h at 1 μM. Scale bars, 5μm. Data are representative of n=3 biological replicates. (C) Western blot images (top) and TRF1 protein levels (bottom) of h676 cells treated with the indicated compounds for 24h at 1 μM. Data are representative of n=3 (PLKi, HSP90i and RTKi) and n=4 (AuroraI, mTOR, CDKi, Docetaxel, Gemcitabine, ERKi, MEKi) biological replicates.


Figure 2Representative images ( left ) and percentage ( right ) of cells presenting 1 or more γH2AX and RAP1 colocalizing foci ( TIFs ) upon treatment of CHA9 - 3 lung cancer cells with the indicated compounds . White arrowheads point to colocalization of γH2AX and RAP1 . Scale bars , 5μm . Data are representative of n = 6 ( DMSO ) and n = 3 ( mTORi , PI3Ki , RTKi , MEKi , ERKi , HSPO90i , CDKi , Docetaxel ) biological replicatesRepresentative images ( left ) and percentage ( right ) of γH2AX - positive cells per field in DMSO or compound treated patient - derived h676 GSCs . Scale bars , 50μm . Data are representative of 6 ( DMSO ) and 3 ( mTORi , Docetaxel , ERKi , MEKi , RTKi , HSP90i , Gemcitabine , CDKi ) biological replicatesQuantification of multitelomeric signals ( MTS ) in h676 GSC metaphases upon treatment with the indicated compounds . Representative images of the qFISH in the metaphases ( left ) . Multitelomeric signals are indicated by arrowheads . Scale bars , 1μm . Data are representative of n = 31 ( DMSO ) , n = 18 ( mTORi ) , n = 11 ( MEKi ) and n = 24 ( RTKi ) biological replicates . Data are represented as mean ± SEM . Significant differences using unpaired t ‐ test are indicated by * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Representative images (left) and percentage (right) of cells presenting 1 or more γH2AX and RAP1 colocalizing foci (TIFs) upon treatment of CHA9-3 lung cancer cells with the indicated compounds. White arrowheads point to colocalization of γH2AX and RAP1. Scale bars, 5μm. Data are representative of n=6 (DMSO) and n=3 (mTORi, PI3Ki, RTKi, MEKi, ERKi, HSPO90i, CDKi, Docetaxel) biological replicatesRepresentative images (left) and percentage (right) of γH2AX-positive cells per field in DMSO or compound treated patient-derived h676 GSCs. Scale bars, 50μm. Data are representative of 6 (DMSO) and 3 (mTORi, Docetaxel, ERKi, MEKi, RTKi, HSP90i, Gemcitabine, CDKi) biological replicatesQuantification of multitelomeric signals (MTS) in h676 GSC metaphases upon treatment with the indicated compounds. Representative images of the qFISH in the metaphases (left). Multitelomeric signals are indicated by arrowheads. Scale bars, 1μm. Data are representative of n=31 (DMSO), n=18 (mTORi), n=11 (MEKi) and n=24 (RTKi) biological replicates. Data are represented as mean ± SEM. Significant differences using unpaired t‐test are indicated by *P < 0.05, **P < 0.01, ***P < 0.001.


Figure 3Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations . Data are representative of n = 2 biological replicates . Data are represented as mean ± SEM normalized to DMSO . Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations . Data are representative of n = 2 biological replicates . Data are represented as mean ± SEM normalized to DMSO . Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations . Data are representative of n = 2 biological replicates . Data are represented as mean ± SEM normalized to DMSO . Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations . Data are representative of n = 2 biological replicates . Data are represented as mean ± SEM normalized to DMSO . Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations . Data are representative of n = 2 biological replicates . Data are represented as mean ± SEM normalized to DMSO . Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations . Data are representative of n = 2 biological replicates . Data are represented as mean ± SEM normalized to DMSO . Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations . Data are representative of n = 2 biological replicates . Data are represented as mean ± SEM normalized to DMSO . Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations . Data are representative of n = 2 biological replicates . Data are represented as mean ± SEM normalized to DMSO .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM normalized to DMSO.Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM normalized to DMSO.Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM normalized to DMSO.Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM normalized to DMSO.Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM normalized to DMSO.Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM normalized to DMSO.Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM normalized to DMSO.Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM normalized to DMSO.


Figure 41 or 2 μM of GST or GST - TRF1 were incubated with the indicated concentrations of mouse ERK2 kinase in the presence of 5μCi [ γ - 32 - P ] ATP . The mixture was resolve by SDS - PAGE followed by autoradiography . 1 or 2 μM of GST or GST - TRF1 were incubated with the indicated concentrations of human b - Raf kinase ( WT or V600E ) in the presence of 5μCi [ γ - 32 - P ] ATP . The mixture was resolve by SDS - PAGE followed by autoradiography . 1 or 2 μM of GST or GST - TRF1 were incubated with the indicated concentrations of mouse MEK1 kinase in the presence of 5μCi [ γ - 32 - P ] ATP . The mixture was resolve by SDS - PAGE followed by autoradiography . 1 μM of GST - TRF1 and 0 . 2 μM of mouse ERK2 kinase were incubated in the presence of ERK and MEK inhibitors . 2 μM of GST - TRF1 and 0 . 1 μM of human b - Raf kinase were incubated in the presence of the bRaf inhibitors dabrafenib and vemurafenib . 1 or 2 μM of GST or GST - TRF1 were incubated with the indicated concentrations of human mTOR kinase1 or 2 μM of GST or GST - TRF1 were incubated with the indicated concentrations of human mTOR kinase in the presence of the mTOR inhibitors rapamycin and Ku0063794Phosphopeptide peak intensity normalized to total TRF1 signal in samples containing only TRF1 or TRF1 plus ERK2Phosphopeptide peak intensity normalized to total TRF1 signal in samples containing only TRF1 plus bRAFWT or bRAFV600EPhosphopeptide peak intensity normalized to total TRF1 signal in samples containing only TRF1 plus mTORRepresentative image ( down ) and quantification ( up ) of in vitro phosphorylation assays with the indicated GST - TRF1 wild - type or mutated forms in the presence of mouse ERK2 kinase .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 41 or 2 μM of GST or GST-TRF1 were incubated with the indicated concentrations of mouse ERK2 kinase in the presence of 5μCi [γ-32-P]ATP. The mixture was resolve by SDS-PAGE followed by autoradiography.1 or 2 μM of GST or GST-TRF1 were incubated with the indicated concentrations of human b-Raf kinase (WT or V600E) in the presence of 5μCi [γ-32-P]ATP. The mixture was resolve by SDS-PAGE followed by autoradiography.1 or 2 μM of GST or GST-TRF1 were incubated with the indicated concentrations of mouse MEK1 kinase in the presence of 5μCi [γ-32-P]ATP. The mixture was resolve by SDS-PAGE followed by autoradiography.1 μM of GST-TRF1 and 0.2 μM of mouse ERK2 kinase were incubated in the presence of ERK and MEK inhibitors.2 μM of GST-TRF1 and 0.1 μM of human b-Raf kinase were incubated in the presence of the bRaf inhibitors dabrafenib and vemurafenib.1 or 2 μM of GST or GST-TRF1 were incubated with the indicated concentrations of human mTOR kinase1 or 2 μM of GST or GST-TRF1 were incubated with the indicated concentrations of human mTOR kinase in the presence of the mTOR inhibitors rapamycin and Ku0063794Phosphopeptide peak intensity normalized to total TRF1 signal in samples containing only TRF1 or TRF1 plus ERK2Phosphopeptide peak intensity normalized to total TRF1 signal in samples containing only TRF1 plus bRAFWT or bRAFV600EPhosphopeptide peak intensity normalized to total TRF1 signal in samples containing only TRF1 plus mTORRepresentative image (down) and quantification (up) of in vitro phosphorylation assays with the indicated GST-TRF1 wild-type or mutated forms in the presence of mouse ERK2 kinase.


Figure 5Western blot images of Trf1lox / lox MEFS with or without overexpression of eGFP - Trf1 WT or mutant alleles followed by Cre recombinase transduction . Quantification of eGFP - TRF1 inhibition in Trf1Δ / Δ MEFs transduced with eGFP - Trf1 WT or mutant alleles as indicated . Data are representative of n = 15 biological replicatesGrowth curves of Trf1Δ / Δ MEFs transduced with eGFP - Trf1 WT or mutant alleles as indicated . Data are representative of n = 5 biological replicatesWestern blot images of Trf1lox / lox MEFS with or without overexpression of eGFP - Trf1 WT or mutant alleles followed by Cre recombinase transduction . Quantification of eGFP - TRF1 inhibition in Trf1Δ / Δ MEFs transduced with eGFP - Trf1 WT or mutant alleles as indicated . Data are representative of n = 5 biological replicatesWestern blot images of p53 - / - MEFS with or without overexpression of eGFP - Trf1 WT or mutant alleles followed treatment with ERKi . Data are representative of n = 2 biological replicatesRepresentative images ( above ) and percentage ( bottom ) of Telomeric and 53BP1 colocalizing foci ( TIFs ) per cells of p53 - / - MEFS with or without overexpression of eGFP - Trf1 WT and the indicated mutants upon treatment with the ERKi . White arrowheads : colocalization of telomeric and 53BP1 . Scale bars , 10μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Western blot images of Trf1lox/lox MEFS with or without overexpression of eGFP-Trf1 WT or mutant alleles followed by Cre recombinase transduction.Quantification of eGFP-TRF1 inhibition in Trf1Δ/Δ MEFs transduced with eGFP-Trf1 WT or mutant alleles as indicated. Data are representative of n=15 biological replicatesGrowth curves of Trf1Δ/Δ MEFs transduced with eGFP-Trf1 WT or mutant alleles as indicated. Data are representative of n=5 biological replicatesWestern blot images of Trf1lox/lox MEFS with or without overexpression of eGFP-Trf1 WT or mutant alleles followed by Cre recombinase transduction. Quantification of eGFP-TRF1 inhibition in Trf1Δ/Δ MEFs transduced with eGFP-Trf1 WT or mutant alleles as indicated. Data are representative of n=5 biological replicatesWestern blot images of p53-/- MEFS with or without overexpression of eGFP-Trf1 WT or mutant alleles followed treatment with ERKi. Data are representative of n=2 biological replicatesRepresentative images (above) and percentage (bottom) of Telomeric and 53BP1 colocalizing foci (TIFs) per cells of p53-/- MEFS with or without overexpression of eGFP-Trf1 WT and the indicated mutants upon treatment with the ERKi. White arrowheads: colocalization of telomeric and 53BP1. Scale bars, 10μm.


Figure 6Western blot image ( above ) and quantification ( bottom ) of TRF1 protein levels upon genetic depletion of ERK1 / 2 in p53 - / - MEF line . Data are representative of n = 3 independent experiments . Representative images ( above ) and quantification ( bottom ) of TRF1 telomeric foci in ERK1 / 2 RNA interfered p53 - / - MEFs . Scale bars , 5μm . Data are representative of n = 2 independent experiments . Western blot image of eGFP - tagged and endogenous TRF1 protein levels upon genetic depletion of ERK1 / 2 in p53 - / - MEF line . Representative images ( above ) and percentage ( bottom ) of telomeric and 53BP1 colocalizing foci ( TIFs ) per cell in p53 - / - MEF with or without overexpression of eGFP - Trf1 WT and indicated mutants upon genetic depletion of ERK1 / 2 . White arrowheads : colocalization of telomeric and 53BP1 . Scale bars , 10μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Western blot image (above) and quantification (bottom) of TRF1 protein levels upon genetic depletion of ERK1/2 in p53-/- MEF line. Data are representative of n=3 independent experiments.Representative images (above) and quantification (bottom) of TRF1 telomeric foci in ERK1/2 RNA interfered p53-/- MEFs. Scale bars, 5μm. Data are representative of n=2 independent experiments.Western blot image of eGFP-tagged and endogenous TRF1 protein levels upon genetic depletion of ERK1/2 in p53-/- MEF line.Representative images (above) and percentage (bottom) of telomeric and 53BP1 colocalizing foci (TIFs) per cell in p53-/- MEF with or without overexpression of eGFP-Trf1 WT and indicated mutants upon genetic depletion of ERK1/2. White arrowheads: colocalization of telomeric and 53BP1.Scale bars, 10μm.


Figure 7Representative images ( left ) and quantification ( right ) of number of spheres formed by h676 GSCs 7 days after treatment with the indicated compounds as single agents or in combination . Scale bars , 100μm . Data are representative of n = 6 biological replicates . Representative images ( left ) and quantification ( right ) of number of spheres formed by h676 GSCs 7 days after treatment with the indicated compounds as single agents or in combination . Scale bars , 100μm . Data are representative of n = 6 biological replicates . Representative images ( left ) and quantification ( right ) of number of spheres formed by h676 GSCs 7 days after treatment with the indicated compounds as single agents or in combination . Scale bars , 100μm . Data are representative of n = 6 biological replicates . Representative images ( left ) and quantification ( right ) of number of spheres formed by h676 GSCs 7 days after treatment with the indicated compounds as single agents or in combination . Scale bars , 100μm . Data are representative of n = 6 biological replicates . Representative images ( left ) and quantification ( right ) of number of spheres formed by h676 GSCs 7 days after treatment with the indicated compounds as single agents or in combination . Scale bars , 100μm . Data are representative of n = 6 biological replicates . Representative images ( left ) and quantification ( right ) of number of spheres formed by h676 GSCs 7 days after treatment with the indicated compounds as single agents or in combination . Scale bars , 100μm . Data are representative of n = 6 biological replicates . Western blot images ( left ) and TRF1 protein levels ( right ) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination . Data are representative of n = 3 ( RTKi in G ( DMSO in GWestern blot images ( left ) and TRF1 protein levels ( right ) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination . Data are representative of ( DMSO in and ERKi in H ) Western blot images ( left ) and TRF1 protein levels ( right ) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination . Data are representative of ( DMSO in ( PI3Ki inWestern blot images ( left ) and TRF1 protein levels ( right ) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination . Data are representative of ( DMSO in ( PI3Ki inWestern blot images ( left ) and TRF1 protein levels ( right ) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination . Data are representative of ( DMSOWestern blot images ( left ) and TRF1 protein levels ( right ) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination . Data are representative of Docetaxel (

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Representative images (left) and quantification (right) of number of spheres formed by h676 GSCs 7 days after treatment with the indicated compounds as single agents or in combination. Scale bars, 100μm. Data are representative of n=6 biological replicates.Representative images (left) and quantification (right) of number of spheres formed by h676 GSCs 7 days after treatment with the indicated compounds as single agents or in combination. Scale bars, 100μm. Data are representative of n=6 biological replicates.Representative images (left) and quantification (right) of number of spheres formed by h676 GSCs 7 days after treatment with the indicated compounds as single agents or in combination. Scale bars, 100μm. Data are representative of n=6 biological replicates.Representative images (left) and quantification (right) of number of spheres formed by h676 GSCs 7 days after treatment with the indicated compounds as single agents or in combination. Scale bars, 100μm. Data are representative of n=6 biological replicates.Representative images (left) and quantification (right) of number of spheres formed by h676 GSCs 7 days after treatment with the indicated compounds as single agents or in combination. Scale bars, 100μm. Data are representative of n=6 biological replicates.Representative images (left) and quantification (right) of number of spheres formed by h676 GSCs 7 days after treatment with the indicated compounds as single agents or in combination. Scale bars, 100μm. Data are representative of n=6 biological replicates.Western blot images (left) and TRF1 protein levels (right) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination. Data are representative of n=3 (RTKi in G (DMSO in GWestern blot images (left) and TRF1 protein levels (right) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination. Data are representative of (DMSO in and ERKi in H)Western blot images (left) and TRF1 protein levels (right) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination. Data are representative of (DMSO in (PI3Ki inWestern blot images (left) and TRF1 protein levels (right) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination. Data are representative of (DMSO in (PI3Ki inWestern blot images (left) and TRF1 protein levels (right) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination. Data are representative of (DMSOWestern blot images (left) and TRF1 protein levels (right) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination. Data are representative of Docetaxel (DMSO


Figure 8Longitudinal tumor growth follow - up in mice injected with h676 GSCs and treated with the indicated compounds in single agents or combination . n represents number of tumors , in A : vehicle n = 16 , PI3Ki n = 8 , ERKi n = 16 , Combination n = 8 Pvalues represent the mean of all the time pointsLongitudinal tumor growth follow - up in mice injected with h676 GSCs and treated with the indicated compounds in single agents or combination . n represents number of tumors , in B : Vehicle n = 16 , PI3Ki n = 16 , MEKi n = 8 , Combination n = 8 Pvalues represent the mean of all the time pointsLongitudinal tumor growth follow - up in mice injected with h676 GSCs and treated with the indicated compounds in single agents or combination . n represents number of tumors , in C : Vehicle n = 8 , PI3Ki n = 16 , Docetaxel n = 16 , Combination n = 8 Pvalues represent the mean of all the time pointsLongitudinal tumor growth follow - up in mice injected with h676 GSCs and treated with the indicated compounds in single agents or combination . n represents number of tumors , in D : Vehicle n = 16 , PI3K n = 16 , Gemcitabine n = 4 , Combination n = 4 . Pvalues represent the mean of all the time pointsRepresentative images ( top ) and quantification ( bottom ) of TRF1 nuclear fluorescence in tumors treated with the indicated compounds as single agents or in combination . Scale bars , 10 μm . Data are represented as mean ± SEM . n represents number of tumors : in E : vehicle n = 15 , PI3Ki n = 10 , ERKi n = 8 , Combination n = 2Representative images ( top ) and quantification ( bottom ) of TRF1 nuclear fluorescence in tumors treated with the indicated compounds as single agents or in combination . Scale bars , 10 μm . Data are represented as mean ± SEM . n represents number of tumors in F : Vehicle n = 15 , PI3Ki n = 10 , MEKi n = 8 , Combination n = 6Representative images ( top ) and quantification ( bottom ) of TRF1 nuclear fluorescence in tumors treated with the indicated compounds as single agents or in combination . Scale bars , 10 μm . Data are represented as mean ± SEM . n represents number of tumors ; in G : Vehicle n = 15 , PI3Ki n = 10 , Docetaxel n = 8 , Combination n = 4Representative images ( top ) and quantification ( bottom ) of TRF1 nuclear fluorescence in tumors treated with the indicated compounds as single agents or in combination . Scale bars , 10 μm . Data are represented as mean ± SEM . n represents number of tumors ; in H : Vehicle n = 15 , PI3K n = 10 , Gemcitabine n = 8 , Combination n = 6 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8Longitudinal tumor growth follow-up in mice injected with h676 GSCs and treated with the indicated compounds in single agents or combination. n represents number of tumors, in A: vehicle n=16, PI3Ki n=8, ERKi n=16, Combination n=8 Pvalues represent the mean of all the time pointsLongitudinal tumor growth follow-up in mice injected with h676 GSCs and treated with the indicated compounds in single agents or combination. n represents number of tumors, in B: Vehicle n=16, PI3Ki n=16, MEKi n=8, Combination n=8 Pvalues represent the mean of all the time pointsLongitudinal tumor growth follow-up in mice injected with h676 GSCs and treated with the indicated compounds in single agents or combination. n represents number of tumors, in C: Vehicle n=8, PI3Ki n=16, Docetaxel n=16, Combination n=8 Pvalues represent the mean of all the time pointsLongitudinal tumor growth follow-up in mice injected with h676 GSCs and treated with the indicated compounds in single agents or combination. n represents number of tumors, in D: Vehicle n=16, PI3K n=16, Gemcitabine n=4, Combination n=4. Pvalues represent the mean of all the time pointsRepresentative images (top) and quantification (bottom) of TRF1 nuclear fluorescence in tumors treated with the indicated compounds as single agents or in combination. Scale bars, 10 μm. Data are represented as mean ± SEM. n represents number of tumors: in E: vehicle n=15, PI3Ki n=10, ERKi n=8, Combination n=2Representative images (top) and quantification (bottom) of TRF1 nuclear fluorescence in tumors treated with the indicated compounds as single agents or in combination. Scale bars, 10 μm. Data are represented as mean ± SEM. n represents number of tumors in F: Vehicle n=15, PI3Ki n=10, MEKi n=8, Combination n=6Representative images (top) and quantification (bottom) of TRF1 nuclear fluorescence in tumors treated with the indicated compounds as single agents or in combination. Scale bars, 10 μm. Data are represented as mean ± SEM. n represents number of tumors ; in G: Vehicle n=15, PI3Ki n=10, Docetaxel n=8, Combination n=4Representative images (top) and quantification (bottom) of TRF1 nuclear fluorescence in tumors treated with the indicated compounds as single agents or in combination. Scale bars, 10 μm. Data are represented as mean ± SEM. n represents number of tumors ; in H: Vehicle n=15, PI3K n=10, Gemcitabine n=8, Combination n=6.


Figure 1B ) Analysis of puromycin incorporation in MelJuSo cells that were left untreated ( - HS ) , exposed to 43 ° C for 30 minutes ( + HS ) , and followed for 4 hours after heat shock ( HS + 4h Rec . ) . The blots were probed with antibodies against puromycin , and GAPDH as loading control . C ) Representative images of MelJuSo cells expressing Ub - YFP , which were either left untreated ( - HS ) , exposed to 43 ° C for 30 minutes ( + HS ) , and followed for 4 hours after heat shock ( HS + 4h Rec . ) .

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Figure 1B) Analysis of puromycin incorporation in MelJuSo cells that were left untreated (- HS), exposed to 43°C for 30 minutes (+ HS), and followed for 4 hours after heat shock (HS + 4h Rec.). The blots were probed with antibodies against puromycin, and GAPDH as loading control.C) Representative images of MelJuSo cells expressing Ub-YFP, which were either left untreated (- HS), exposed to 43 °C for 30 minutes (+ HS), and followed for 4 hours after heat shock (HS + 4h Rec.).


Figure 2A ) and B ) Representative images of stress granule proficient ( siControl ) and deficient ( siG3BP1 / 2 ) MelJuSo cells expressing the nuclear UPS reporter NLS - GFP - CL1 ( A ) and the cytoplasmic UPS reporter NES - GFP - CL1 ( B ) . The cells were left untreated ( - HS ) , exposed to 43 ° C for 30 minutes ( HS ) , or exposed to 43 ° C for 30 minutes and followed by 4 hours recovery ( HS + 4h Rec . ) . Nucleolar accumulation of NLS - GFP - CL1 during the recovery is indicated by arrows in A ) . C ) and D ) Quantifications of the mean cellular GFP fluorescence intensities of A ) and B ) normalized to untreated control cells ( siControl - HS ) . The frequency and distribution of the relative fluorescence intensities per cell are shown as violin plots . The solid lines in each distribution represent the median , and dash lines represent the upper and lower interquartile range limits . ( n = 3 independent experiments , > 1000 cells analyzed per condition , Kruskal - Wallis test , * * P < 0 . 01 , ns : not significant ) . E ) Representative confocal images of immunofluorescent staining of the nucleolar marker nucleolin and the nuclear UPS reporter NLS - GFP - CL1 in control ( siControl , left panel ) and G3BP1 / 2 - depleted ( siGRBP1 / 2 , right panel ) MelJuSo cells exposed to a 30 min 43 ° C heat shock , followed by 4 hours at 37 ° C ( Recovery ) . Nucleolar accumulation of NLS - GFP - CL1 is indicated by arrows . F ) Quantification of the nucleolus / nucleoplasm ratios of NLS - GFP - CL1 intensities in images of experiment shown in E ) . The frequency and distribution of the ratio per cell are shown as violin plots . The solid lines in each distribution represent the median , and dash lines represent the upper and lower interquartile range limits . ( n = 3 independent experiments , 100 cells analyzed per condition , Kruskal - Wallis test , * * P < 0 . 01 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A) and B) Representative images of stress granule proficient (siControl) and deficient (siG3BP1/2) MelJuSo cells expressing the nuclear UPS reporter NLS-GFP-CL1 (A) and the cytoplasmic UPS reporter NES-GFP-CL1 (B). The cells were left untreated (- HS), exposed to 43°C for 30 minutes (HS), or exposed to 43°C for 30 minutes and followed by 4 hours recovery (HS + 4h Rec.). Nucleolar accumulation of NLS-GFP-CL1 during the recovery is indicated by arrows in A). C) and D) Quantifications of the mean cellular GFP fluorescence intensities of A) and B) normalized to untreated control cells (siControl - HS). The frequency and distribution of the relative fluorescence intensities per cell are shown as violin plots. The solid lines in each distribution represent the median, and dash lines represent the upper and lower interquartile range limits. (n=3 independent experiments, >1000 cells analyzed per condition, Kruskal-Wallis test, ** P<0.01, ns: not significant).E) Representative confocal images of immunofluorescent staining of the nucleolar marker nucleolin and the nuclear UPS reporter NLS-GFP-CL1 in control (siControl, left panel) and G3BP1/2-depleted (siGRBP1/2, right panel) MelJuSo cells exposed to a 30 min 43°C heat shock, followed by 4 hours at 37°C (Recovery). Nucleolar accumulation of NLS-GFP-CL1 is indicated by arrows. F) Quantification of the nucleolus/nucleoplasm ratios of NLS-GFP-CL1 intensities in images of experiment shown in E). The frequency and distribution of the ratio per cell are shown as violin plots. The solid lines in each distribution represent the median, and dash lines represent the upper and lower interquartile range limits. (n=3 independent experiments, 100 cells analyzed per condition, Kruskal-Wallis test, ** P<0.01).


Figure 3A ) Representative confocal images of immunofluorescent staining of the stress granule marker TIA1 and puromycin - labelled proteins in parental and G3BP1 / 2 knock - out U2OS cells . Cells were subjected to 43 ° C heat shock . B ) Quantification of the nucleus / cytoplasm ratios of puromycin intensities in images from A ) . The frequency and distribution of the ratio per cell are shown as violin plots . The solid lines in each distribution represent the median , and dash lines represent the upper and lower interquartile range limits ( n = 3 independent experiments , > 50 cells analyzed per condition , Kruskal - Wallis test , * * P < 0 . 01 ) . E ) Representative confocal images of immunofluorescent staining of the stress granule marker TIA1 and puromycin - labelled proteins in control ( siControl ) and Hsp70 - depleted ( siHsp70 ) U2OS cells . Cells were subjected to 43 ° C heat shock F ) Quantification of the numbers of puromycin positive TIA dots per cell in images from E ) . The numbers of positive foci per cell are shown as violin plots . The solid lines in each distribution represent the median , and dash lines represent the upper and lower interquartile range limits ( n = 3 independent experiments , > 50 cells analyzed per condition , Kruskal - Wallis test , * * P < 0 . 01 ) . G ) Representative confocal images of immunofluorescent staining of the nucleolar marker nucleolin and puromycin - labelled proteins in control ( siControl ) and Hsp70 - depleted ( siHsp70 ) G3BP1 / 2 knock - out U2OS cells . Cells were subjected to 43 ° C heat shock . H ) Quantification of the nucleolus / nucleoplasm ratio of puromycin intensities in images from G ) . The frequency and distribution of the ratio per cell are shown as violin plots . The solid lines in each distribution represent the median , and dash lines represent the upper and lower interquartile range limits ( n = 3 independent experiments , > 50 cells analyzed per condition , Mann - Whitney test , * * P < 0 . 01 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A) Representative confocal images of immunofluorescent staining of the stress granule marker TIA1 and puromycin-labelled proteins in parental and G3BP1/2 knock-out U2OS cells. Cells were subjected to 43°C heat shock. B) Quantification of the nucleus/cytoplasm ratios of puromycin intensities in images from A). The frequency and distribution of the ratio per cell are shown as violin plots. The solid lines in each distribution represent the median, and dash lines represent the upper and lower interquartile range limits (n=3 independent experiments, >50 cells analyzed per condition, Kruskal-Wallis test, ** P<0.01).E) Representative confocal images of immunofluorescent staining of the stress granule marker TIA1 and puromycin-labelled proteins in control (siControl) and Hsp70-depleted (siHsp70) U2OS cells. Cells were subjected to 43°C heat shock F) Quantification of the numbers of puromycin positive TIA dots per cell in images from E). The numbers of positive foci per cell are shown as violin plots. The solid lines in each distribution represent the median, and dash lines represent the upper and lower interquartile range limits (n=3 independent experiments, >50 cells analyzed per condition, Kruskal-Wallis test, ** P<0.01).G) Representative confocal images of immunofluorescent staining of the nucleolar marker nucleolin and puromycin-labelled proteins in control (siControl) and Hsp70-depleted (siHsp70) G3BP1/2 knock-out U2OS cells. Cells were subjected to 43°C heat shock. H) Quantification of the nucleolus/nucleoplasm ratio of puromycin intensities in images from G). The frequency and distribution of the ratio per cell are shown as violin plots. The solid lines in each distribution represent the median, and dash lines represent the upper and lower interquartile range limits (n=3 independent experiments, >50 cells analyzed per condition, Mann-Whitney test, ** P<0.01).


Figure 4B ) Representative confocal images of immunofluorescent staining of HSF1 in parental and G3BP1 / 2 knock - out U2OS cells directly after heat shock ( + HS ) and 2 hours after 2 hours recovery ( HS + 2h Recovery ) . C ) Quantification of the number of HSF1 foci per cell in images from B ) , shown as box plot with median and 5 - 95 percentiles ( n = 3 independent experiments , > 300 cells analyzed per condition , Kruskal - Wallis test , * * P < 0 . 01 ) . D ) Western blot analysis of parental and G3BP1 / 2 knock - out U2OS cells that were left untreated ( - HS ) , heat shocked ( HS ) , and followed for 1 , 2 , and 4 hours after heat shock ( Rec ) . The blots were probed with antibodies against HSF1 and GAPDH ( * indicated phosphorylated HSF1 , and * * indicates unphosphorylated HSF1 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4B) Representative confocal images of immunofluorescent staining of HSF1 in parental and G3BP1/2 knock-out U2OS cells directly after heat shock (+ HS) and 2 hours after 2 hours recovery (HS + 2h Recovery). C) Quantification of the number of HSF1 foci per cell in images from B), shown as box plot with median and 5-95 percentiles (n=3 independent experiments, >300 cells analyzed per condition, Kruskal-Wallis test, ** P<0.01).D) Western blot analysis of parental and G3BP1/2 knock-out U2OS cells that were left untreated (- HS), heat shocked (HS), and followed for 1, 2, and 4 hours after heat shock (Rec). The blots were probed with antibodies against HSF1 and GAPDH (* indicated phosphorylated HSF1, and ** indicates unphosphorylated HSF1).


Figure 5A ) Representative confocal images of immunofluorescent staining of the nucleolar marker nucleolin and SUMO2 / 3 in parental and G3BP1 / 2 knock - out U2OS cells . Cells were subjected to a heat shock followed by 2 hours recovery . B ) Quantification of the nucleolus / nucleoplasm ratio of SUMO2 / 3 intensities in images from A ) . The frequency and distribution of the ratio per cell are shown as violin plots . The solid lines in each distribution represent the median , and dash lines represent the upper and lower interquartile range limits ( n = 3 independent experiments , > 50 cells analyzed per condition , Mann - Whitney test , * * P < 0 . 01 ) . C ) Representative confocal images of immunofluorescent staining of PML bodies ( PML ) and SUMO2 / 3 in parental and G3BP1 / 2 knock - out U2OS cells . Cells were heat shocked followed by 2 hours recovery . Arrows indicate dots positive for PML and SUMO2 / 3 staining . D ) Quantification of SUMO2 / 3 positive PML puncta in images from C ) . Data represent the mean ± SD ( n = 3 independent experiments , > 50 cells analyzed per condition , Mann - Whitney test , * * P < 0 . 01 ) . E ) Analysis of SUMO2 / 3 conjugates in parental and G3BP1 / 2 knock - out U2OS cells that were left untreated ( - HS ) , exposed to a heat shock ( + HS ) , and followed for 2 hours ( 2h Rec . ) with or without 100 nM proteasome inhibitor epoxomicin ( EPX ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A) Representative confocal images of immunofluorescent staining of the nucleolar marker nucleolin and SUMO2/3 in parental and G3BP1/2 knock-out U2OS cells. Cells were subjected to a heat shock followed by 2 hours recovery. B) Quantification of the nucleolus/nucleoplasm ratio of SUMO2/3 intensities in images from A). The frequency and distribution of the ratio per cell are shown as violin plots. The solid lines in each distribution represent the median, and dash lines represent the upper and lower interquartile range limits (n=3 independent experiments, >50 cells analyzed per condition, Mann-Whitney test, ** P<0.01).C) Representative confocal images of immunofluorescent staining of PML bodies (PML) and SUMO2/3 in parental and G3BP1/2 knock-out U2OS cells. Cells were heat shocked followed by 2 hours recovery. Arrows indicate dots positive for PML and SUMO2/3 staining. D) Quantification of SUMO2/3 positive PML puncta in images from C). Data represent the mean ± SD (n=3 independent experiments, >50 cells analyzed per condition, Mann-Whitney test, ** P<0.01).E) Analysis of SUMO2/3 conjugates in parental and G3BP1/2 knock-out U2OS cells that were left untreated (- HS), exposed to a heat shock (+ HS), and followed for 2 hours (2h Rec.) with or without 100 nM proteasome inhibitor epoxomicin (EPX).


Figure 6A ) Representative confocal images of immunofluorescent staining of SUMO2 / 3 and TDP43 in parental and G3BP1 / 2 knock - out U2OS cells . Cells were exposed to a heat shocked followed by 2 hours recovery . Arrows indicate dots positive for TDP - 43 and SUMO2 / 3 staining . B ) Quantification of the percentage of SUMO2 / 3 positive TDP43 puncta in images from experiment shown in A ) . . Data represent the mean ± SD ( n = 3 independent experiments , > 50 cells analyzed per condition , Mann - Whitney test , * * P < 0 . 01 ) . D ) Analysis of detergent SDS and NP40 soluble fractions of TDP43 in parental and G3BP1 / 2 knock - out U2OS cells that were left untreated ( - HS ) , exposed to 43 ° C ( + HS ) followed by 2 hours recovery ( 2h Rec . ) in absence or presence of 100 nM proteasome inhibitor epoxomicin ( EPX ) . E ) Quantification of the TDP43 band densities in the SDS - soluble fraction . Data represent the mean ± SD ( n = 3 independent experiments , Student ' s unpaired t - test , * P < 0 . 05 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A) Representative confocal images of immunofluorescent staining of SUMO2/3 and TDP43 in parental and G3BP1/2 knock-out U2OS cells. Cells were exposed to a heat shocked followed by 2 hours recovery. Arrows indicate dots positive for TDP-43 and SUMO2/3 staining. B) Quantification of the percentage of SUMO2/3 positive TDP43 puncta in images from experiment shown in A)..Data represent the mean ± SD (n=3 independent experiments, >50 cells analyzed per condition, Mann-Whitney test, ** P<0.01).D) Analysis of detergent SDS and NP40 soluble fractions of TDP43 in parental and G3BP1/2 knock-out U2OS cells that were left untreated (-HS), exposed to 43°C (+HS) followed by 2 hours recovery (2h Rec.) in absence or presence of 100 nM proteasome inhibitor epoxomicin (EPX). E) Quantification of the TDP43 band densities in the SDS-soluble fraction. Data represent the mean ± SD (n=3 independent experiments, Student's unpaired t-test, * P<0.05).


Figure 7A ) Analysis of SUMO2 / 3 conjugates in control ( siControl ) or RNF4 - depleted ( siRNF4 ) G3BP1 / 2 knock - out U2OS cells . Cells were left untreated ( - HS ) , exposed to 43 ° C ( + HS ) , followed by 2 hours recovery ( HS + 2h Rec . ) in the absence or presence of 100 nM proteasome inhibitor epoxomicin ( EPX ) . B ) Quantification of the total SUMO2 / 3 band densities . Data represent the mean ± SD ( n = 3 independent experiments , * P < 0 . 05 ) . E ) Representative confocal images of control ( siControl ) , RNF4 - depleted ( siRNF4 ) , G3BP1 / 2 - depleted ( siG3BP1 / 2 ) and G3BP1 / 2 + RNF4 - depleted ( siG3BP1 / 2 + siRNF4 ) MelJuSo cells expressing the nuclear UPS reporter NLS - GFP - CL1 . Cells were exposed to heat shock followed by 4 hours recovery in the absence or presence of the proteasome inhibitor epoxomicin ( EPX ) . The nucleolar marker nucleolin was stained by immunofluorescence .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A) Analysis of SUMO2/3 conjugates in control (siControl) or RNF4-depleted (siRNF4) G3BP1/2 knock-out U2OS cells. Cells were left untreated (-HS), exposed to 43°C (+ HS), followed by 2 hours recovery (HS + 2h Rec.) in the absence or presence of 100 nM proteasome inhibitor epoxomicin (EPX). B) Quantification of the total SUMO2/3 band densities. Data represent the mean ± SD (n=3 independent experiments, *P<0.05).E) Representative confocal images of control (siControl), RNF4-depleted (siRNF4), G3BP1/2-depleted (siG3BP1/2) and G3BP1/2+RNF4-depleted (siG3BP1/2 + siRNF4) MelJuSo cells expressing the nuclear UPS reporter NLS-GFP-CL1. Cells were exposed to heat shock followed by 4 hours recovery in the absence or presence of the proteasome inhibitor epoxomicin (EPX). The nucleolar marker nucleolin was stained by immunofluorescence.


Figure 8A ) Representative confocal images of immunofluorescent staining of FLAG - tagged ubiquitin ( FLAG - Ub ) and mutant ataxin1 ( ATX1 ) in parental and G3BP1 / 2 knock - out U2OS cells . Cells were subjected to heat shock followed by 4 hours recovery . B ) Frequency distribution of ataxin - 1 inclusions per cell from three independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8A) Representative confocal images of immunofluorescent staining of FLAG-tagged ubiquitin (FLAG-Ub) and mutant ataxin1 (ATX1) in parental and G3BP1/2 knock-out U2OS cells. Cells were subjected to heat shock followed by 4 hours recovery. B) Frequency distribution of ataxin-1 inclusions per cell from three independent experiments.