f3 ( a ) Three - colour flow cytometry of erythroid cells from WT embryonic liver ( E14 . 5 ) stained with anti - Ter119 , anti - CD71 and Mitotracker Deep Red . ( Left upper panel ) Ter119 versus CD71 fluorescence . ( Other panels ) FSC versus Mitotracker fluorescence of the R1 ( right upper panel ) , R2 ( left lower panel ) and R3 ( right lower panel ) fractions . ( b , c ) Percentage of cells with a low number of mitochondria ( mitochondrialow cells ) among CD71 − Ter119 + EC . Erythroid cells from the liver ( E14 . 5 and E18 . 5 ) and peripheral blood ( E18 . 5 ) of indicated mice were stained with anti - Ter119 , anti - CD71 and Mitotracker Deep Red . ( b ) Representative histograms of mitochondrial content in CD71 − Ter119 + cells . Numbers indicate the population of mitochondrialow cells . ( c ) The percentage of CD71 − Ter119 + cells without mitochondria was determined by gating the mitochondrialow fraction , as shown in ( b ) ( mean ± s . d . , n = 6 ) . * P0 . 05 versus value of WT ( analysis of variance ( ANOVA ) ) ; # P0 . 05 versus value of Atg5 − / − ( ANOVA ) ; ' NS ' indicates not significant versus value of WT ( ANOVA ) . ( d ) Percent mitochondrialow cells in the CD71 − Ter119 + EC of Atg7F / FCre embryo . Erythroid cells from the liver and peripheral blood of WT and Atg7F / FCre embryo ( E18 . 5 ) were stained with anti - Ter119 , anti - CD71 , and Mitotracker Deep Red ( mean ± s . d . , n = 3 ) . * P0 . 05 versus value of WT ( Student ' s t - test ) . ( e ) Percentage of mitochondrialow cells in Syto16low cells . Liver erythroid cells ( E18 . 5 ) were stained with Syto16 ( DNA ) and Mitotracker Deep Red . Representative dot plots of the mitochondrial content of Syto16low cells are demonstrated in Supplementary Fig . 5 , the percentage of Syto16low cells without mitochondria was determined by gating the mitochondrialow fraction ( mean ± s . d . , n = 6 ) . * P0 . 05 versus value of WT ( ANOVA ) ; # P0 . 05 versus value of Atg5 − / − ( ANOVA ) ; ' NS ' indicates not significant versus value of WT ( ANOVA ) . ( f ) Immunofluorescent analysis of mitochondrial and lysosomal proteins . Liver Ter119 + cells ( E18 . 5 ) of the indicated mice were stained with anti - Tom20 ( mitochondrial marker ) and anti - Lamp2 ( lysosomal marker ) antibodies , and observed by fluorescent microscopy . Scale bar , 2 μm . Green , red , white , and blue indicate Tom20 , Lamp2 , Ter119 and DAPI ( DNA ) , respectively . Mitochondrial and lysosomal markers have separate distributions in Ulk1gt / gt cells and DKO cells .

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f3(a) Three-colour flow cytometry of erythroid cells from WT embryonic liver (E14.5) stained with anti-Ter119, anti-CD71 and Mitotracker Deep Red. (Left upper panel) Ter119 versus CD71 fluorescence. (Other panels) FSC versus Mitotracker fluorescence of the R1 (right upper panel), R2 (left lower panel) and R3 (right lower panel) fractions. (b,c) Percentage of cells with a low number of mitochondria (mitochondrialow cells) among CD71−Ter119+ EC. Erythroid cells from the liver (E14.5 and E18.5) and peripheral blood (E18.5) of indicated mice were stained with anti-Ter119, anti-CD71 and Mitotracker Deep Red.(b) Representative histograms of mitochondrial content in CD71−Ter119+ cells. Numbers indicate the population of mitochondrialow cells.(c) The percentage of CD71−Ter119+ cells without mitochondria was determined by gating the mitochondrialow fraction, as shown in (b) (mean±s.d., n=6). *P0.05 versus value of WT (analysis of variance (ANOVA)); #P0.05 versus value of Atg5−/− (ANOVA); 'NS' indicates not significant versus value of WT (ANOVA).(d) Percent mitochondrialow cells in the CD71−Ter119+ EC of Atg7F/FCre embryo. Erythroid cells from the liver and peripheral blood of WT and Atg7F/FCre embryo (E18.5) were stained with anti-Ter119, anti-CD71, and Mitotracker Deep Red (mean±s.d., n=3). *P0.05 versus value of WT (Student's t-test).(e) Percentage of mitochondrialow cells in Syto16low cells. Liver erythroid cells (E18.5) were stained with Syto16 (DNA) and Mitotracker Deep Red. Representative dot plots of the mitochondrial content of Syto16low cells are demonstrated in Supplementary Fig. 5, the percentage of Syto16low cells without mitochondria was determined by gating the mitochondrialow fraction (mean±s.d., n=6). *P0.05 versus value of WT (ANOVA); #P0.05 versus value of Atg5−/− (ANOVA); 'NS' indicates not significant versus value of WT (ANOVA).(f) Immunofluorescent analysis of mitochondrial and lysosomal proteins. Liver Ter119+ cells (E18.5) of the indicated mice were stained with anti-Tom20 (mitochondrial marker) and anti-Lamp2 (lysosomal marker) antibodies, and observed by fluorescent microscopy. Scale bar, 2 μm. Green, red, white, and blue indicate Tom20, Lamp2, Ter119 and DAPI (DNA), respectively. Mitochondrial and lysosomal markers have separate distributions in Ulk1gt/gt cells and DKO cells.


f4 ( a - f ) Representative EM of WT ( a ) , ATG5 − / − ( b ) , Ulk1gt / gt ( c , d ) and DKO ( e , f ) reticulocytes from embryonic liver ( E14 . 5 ) . ( a , b ) Mitophagy was detected in WT and ATG5 − / − cells ( scale bar , 1 μm ) . Insets indicate mitophagy ( scale bar , 0 . 2 μm ) . ( c , e ) In Ulk1gt / gt and DKO cells , numerous mitochondria were in contact with membrane structures ( scale bar , 1 μm ) . Enlarged images are shown on the right side ( scale bar , 0 . 2 μm ) . ( d , f ) Some Ulk1gt / gt and DKO cells showed plasma membrane blebs containing mitochondria ( scale bar , 1 μm ) . ( g , h ) Quantitative analysis of mitophagy calculated from EM photos in the embryonic liver at E18 . 5 . Population of reticulocytes with mitophagy ( g ) , and number of mitochondria per reticulocytes ( h ) were calculated ( n > 35 cells per mouse ) . The data are shown as mean ± s . d . ( n = 3 ) . * P0 . 05 versus value of WT ( analysis of variance ( ANOVA ) ) ; # P0 . 05 versus value of Atg5 − / − ( ANOVA ) ; ' NS ' indicates not significant versus value of WT ( ANOVA ) .

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f4(a-f) Representative EM of WT (a), ATG5−/− (b), Ulk1gt/gt (c,d) and DKO (e,f) reticulocytes from embryonic liver (E14.5). (a,b) Mitophagy was detected in WT and ATG5−/− cells (scale bar, 1 μm). Insets indicate mitophagy (scale bar, 0.2 μm). (c,e) In Ulk1gt/gt and DKO cells, numerous mitochondria were in contact with membrane structures (scale bar, 1 μm). Enlarged images are shown on the right side (scale bar, 0.2 μm). (d,f) Some Ulk1gt/gt and DKO cells showed plasma membrane blebs containing mitochondria (scale bar, 1 μm). (g,h) Quantitative analysis of mitophagy calculated from EM photos in the embryonic liver at E18.5. Population of reticulocytes with mitophagy (g), and number of mitochondria per reticulocytes (h) were calculated (n>35 cells per mouse). The data are shown as mean±s.d. (n=3). *P0.05 versus value of WT (analysis of variance (ANOVA)); #P0.05 versus value of Atg5−/− (ANOVA); 'NS' indicates not significant versus value of WT (ANOVA).


f5 ( a ) Erythroblasts ( EB ; mitochondriahigh Syto16high ) , reticulocytes ( RC ; mitochondriahigh Syto16low ) and EC ( EC ; Ter119 + PBC ) were collected from the indicated embryos ( E18 . 5 ) , and the expression of each protein was examined by western blot . The asterisk indicates a nonspecific band . Uncropped images are shown in Supplementary Figs 11 and 12 . ( b ) Semi - quantitative analysis of the expression level of each protein and the LC3 - II / LC3 - I ratio ( mean ± s . d . , n = 3 ) . * P0 . 05 versus value of WT EC ( analysis of variance ( ANOVA ) ) ; # P0 . 05 versus value of Atg5 − / − EC ( ANOVA ) ; ' NS ' indicates not significant versus value of WT EC ( ANOVA ) . ( c ) Modification of Ulk1 and Atg13 during erythrocyte maturation . Cell lysates were treated with λ - phosphatase ( 400 U ) for 45 min at 30 ° C , and band shift was examined by anti - Ulk1 and anti - Atg13 antibodies . Asterisk indicates non - specific band . Uncropped images are shown in Supplementary Fig . 12 .

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f5(a) Erythroblasts (EB; mitochondriahigh Syto16high), reticulocytes (RC; mitochondriahigh Syto16low) and EC (EC; Ter119+ PBC) were collected from the indicated embryos (E18.5), and the expression of each protein was examined by western blot. The asterisk indicates a nonspecific band. Uncropped images are shown in Supplementary Figs 11 and 12.(b) Semi-quantitative analysis of the expression level of each protein and the LC3-II/LC3-I ratio (mean±s.d., n=3). *P0.05 versus value of WT EC (analysis of variance (ANOVA)); #P0.05 versus value of Atg5−/− EC (ANOVA); 'NS' indicates not significant versus value of WT EC (ANOVA).(c) Modification of Ulk1 and Atg13 during erythrocyte maturation. Cell lysates were treated with λ-phosphatase (400 U) for 45 min at 30 °C, and band shift was examined by anti-Ulk1 and anti-Atg13 antibodies. Asterisk indicates non-specific band. Uncropped images are shown in Supplementary Fig. 12.


f6 ( a ) Morphology of cultured erythroid cells on day 0 , 1 and 3 of differentiation . Erythroblasts were purified from embryonic liver ( E14 . 5 ) and incubated for the indicated times in erythrocyte differentiation medium . The cells were stained with a Diff - quick kit . ( b ) Percentage of cells without nuclei ( Syto16low cells ) on the indicated days of differentiation ( mean ± s . d . , n = 6 ) . Loss of nuclei occurred normally in all mice . ( c , d ) Percentage of Syto16low cells without mitochondria . Differentiated cells were stained with Syto16 and Mitotracker Deep Red . Representative histograms of mitochondrial content in Syto16low cells on day 6 are shown in ( c ) . Numbers indicate the population of mitochondrialow cells . Representative dot plots are also available in Supplementary Fig . 13 . ( d ) Percentage of Syto16low cells lacking mitochondria determined by gating the mitochondrialow fraction as shown in ( c ) ( mean ± s . d . , n = 6 ) . * P0 . 05 versus value of WT ( analysis of variance ( ANOVA ) ) ; # P0 . 05 versus value of Atg5 − / − ( ANOVA ) ; ' NS ' indicates not significant versus value of WT ( ANOVA ) . ( e - h ) Impact of various drugs on mitochondrial clearance during in vitro differentiation . Differentiated cells from WT embryos were incubated with or without wortmannin ( e ) , 3 - methyladenine ( 3 - MA ) ( f ) , rapamycin ( g ) or compound C ( h ) at the indicated concentrations and time periods . Percentage of mitochondrialow cells / Syto16low cells was measured by flow cytometry ( mean ± s . d . , n = 3 ) . * P0 . 05 versus value of no drug ( ANOVA ) . ( i ) Impact of various drugs on mitochondrial clearance during in vitro differentiation . with cells from Atg5 − / − embryos exposed to wortmannin or 3 - MA ( mean ± s . d . , n = 3 ) . * P0 . 05 versus value of no drug ( ANOVA ) .

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 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 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

f6(a) Morphology of cultured erythroid cells on day 0, 1 and 3 of differentiation. Erythroblasts were purified from embryonic liver (E14.5) and incubated for the indicated times in erythrocyte differentiation medium. The cells were stained with a Diff-quick kit. (b) Percentage of cells without nuclei (Syto16low cells) on the indicated days of differentiation (mean±s.d., n=6). Loss of nuclei occurred normally in all mice.(c,d) Percentage of Syto16low cells without mitochondria. Differentiated cells were stained with Syto16 and Mitotracker Deep Red. Representative histograms of mitochondrial content in Syto16low cells on day 6 are shown in (c). Numbers indicate the population of mitochondrialow cells. Representative dot plots are also available in Supplementary Fig. 13. (d) Percentage of Syto16low cells lacking mitochondria determined by gating the mitochondrialow fraction as shown in (c) (mean±s.d., n=6). *P0.05 versus value of WT (analysis of variance (ANOVA)); #P0.05 versus value of Atg5−/− (ANOVA); 'NS' indicates not significant versus value of WT (ANOVA).(e-h) Impact of various drugs on mitochondrial clearance during in vitro differentiation. Differentiated cells from WT embryos were incubated with or without wortmannin (e), 3-methyladenine (3-MA) (f), rapamycin (g) or compound C (h) at the indicated concentrations and time periods. Percentage of mitochondrialow cells/Syto16low cells was measured by flow cytometry (mean±s.d., n=3). *P0.05 versus value of no drug (ANOVA).(i)Impact of various drugs on mitochondrial clearance during in vitro differentiation.with cells from Atg5−/− embryos exposed to wortmannin or 3-MA (mean±s.d., n=3). *P0.05 versus value of no drug (ANOVA).


f7 ( a ) Number of EC and haemoglobin content in the blood of the indicated mice ( 10 - 20 weeks old ; mean ± s . d . , n = 6 ) . * P0 . 05 versus value of WT ( Student ' s t - test ) . ( b ) Analysis of early erythrocyte development . Bone marrow cells were harvested from the indicated mice ( 10 - 20 weeks old ) and stained with anti - cKit and anti - Sca1 antibodies . The population of each cell type was counted by FACS ( mean ± s . d . , n = 3 ) . * P0 . 05 versus value of WT ( analysis of variance ( ANOVA ) ) . ( c ) Percentage of mitochondria - containing Ter119 + blood cells . Cells in peripheral blood from the indicated mice ( 10 - 20 weeks old ) were stained with anti - Ter119 antibody together with Mitotracker Deep Red . The population of mitochondriahigh cells among Ter119 - positive cells was counted by FACS ( mean ± s . d . , n = 3 ) . * P0 . 05 versus value of WT ( ANOVA ) . ( d ) Percentage of mitochondria - containing Syto16low blood cells . Cells in peripheral blood from the indicated mice ( 10 - 20 weeks old ) were stained with Syto16 and Mitotracker Deep Red . The population of mitochondriahigh cells among Syto16 - negative cells was counted by FACS ( mean ± s . d . , n = 3 ) . * P0 . 05 versus value of WT ( ANOVA ) . ( e ) Representative electron micrographs of bone marrow reticulocytes in 10 week - old WT and Ulk1gt / gt mice ( scale bar , 1 μm ) . Insets indicate mitophagy ( scale bar , 0 . 2 μm ) . ( f , g ) Erythroblasts ( EB ; mitochondriahigh Syto16high ) , reticulocytes ( RC ; mitochondriahigh Syto16low ) and EC ( EC ; Ter119 + PBC ) were collected from the indicated mice ( 10 - 20 weeks old ) , and the expression of each protein was examined by western blot . Uncropped images are shown in Supplementary Fig . 14 . ( g ) Semi - quantitative analysis of the expression level of each protein ( mean ± s . d . , n = 3 ) . ' NS ' indicates not significant versus value of WT EC ( ANOVA ) . ( h , i ) The impact of Ulk1 on mitochondrial clearance during stress erythropoiesis . ( h ) Representative images of erythrocyte in PHZ - treated WT and Ulk1gt / gt mice ( 14 weeks old ) . Blood cells were stained with anti - Ter119 ( erythroid cell marker ) and anti - Tom20 ( mitochondrial marker ) antibodies and observed by fluorescence microscopy . Green and red indicate Ter119 and Tom20 , respectively . Scale bar , 10 μm . ( i ) Percentage of mitochondrialow cells among Syto16low cells . Erythroid cells were stained with Syto16 ( DNA ) and Mitotracker Deep Red ( mean ± s . d . , n = 3 ) . * P0 . 05 versus value of WT ( Student ' s t - test ) . Mitochondrial retention was observed in Ulk1gt / gt mice .

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f7(a) Number of EC and haemoglobin content in the blood of the indicated mice (10-20 weeks old; mean±s.d., n=6). *P0.05 versus value of WT (Student's t-test).(b) Analysis of early erythrocyte development. Bone marrow cells were harvested from the indicated mice (10-20 weeks old) and stained with anti-cKit and anti-Sca1 antibodies. The population of each cell type was counted by FACS (mean±s.d., n=3). *P0.05 versus value of WT (analysis of variance (ANOVA)).(c) Percentage of mitochondria-containing Ter119+ blood cells. Cells in peripheral blood from the indicated mice (10-20 weeks old) were stained with anti-Ter119 antibody together with Mitotracker Deep Red. The population of mitochondriahigh cells among Ter119-positive cells was counted by FACS (mean±s.d., n=3). *P0.05 versus value of WT (ANOVA).(d) Percentage of mitochondria-containing Syto16low blood cells. Cells in peripheral blood from the indicated mice (10-20 weeks old) were stained with Syto16 and Mitotracker Deep Red. The population of mitochondriahigh cells among Syto16-negative cells was counted by FACS (mean±s.d., n=3). *P0.05 versus value of WT (ANOVA).(e) Representative electron micrographs of bone marrow reticulocytes in 10 week-old WT and Ulk1gt/gt mice (scale bar, 1 μm). Insets indicate mitophagy (scale bar, 0.2 μm).(f,g) Erythroblasts (EB; mitochondriahigh Syto16high), reticulocytes (RC; mitochondriahigh Syto16low) and EC (EC; Ter119+ PBC) were collected from the indicated mice (10-20 weeks old), and the expression of each protein was examined by western blot. Uncropped images are shown in Supplementary Fig. 14. (g) Semi-quantitative analysis of the expression level of each protein (mean±s.d., n=3). 'NS' indicates not significant versus value of WT EC (ANOVA).(h,i) The impact of Ulk1 on mitochondrial clearance during stress erythropoiesis. (h) Representative images of erythrocyte in PHZ-treated WT and Ulk1gt/gt mice (14 weeks old). Blood cells were stained with anti-Ter119 (erythroid cell marker) and anti-Tom20 (mitochondrial marker) antibodies and observed by fluorescence microscopy. Green and red indicate Ter119 and Tom20, respectively. Scale bar, 10 μm. (i) Percentage of mitochondrialow cells among Syto16low cells. Erythroid cells were stained with Syto16 (DNA) and Mitotracker Deep Red (mean±s.d., n=3). *P0.05 versus value of WT (Student's t-test). Mitochondrial retention was observed in Ulk1gt/gt mice.


Figure 3A . STAT3 immunohistochemistry - staining of low STAT3 and high STAT3 PCa samples . Red arrows indicate transformed PCa glands , black arrows indicate pre - transformed normal prostate glands . Scale bar = 100µm . # = sample - IDs .

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Figure 3A. STAT3 immunohistochemistry- staining of low STAT3 and high STAT3 PCa samples. Red arrows indicate transformed PCa glands, black arrows indicate pre-transformed normal prostate glands. Scale bar = 100µm. # = sample-IDs.


Figure 4A . Differentially expressed proteins in PtenStat3pc - / - vs . Ptenpc - / - proteomic samples . Colors indicate adj . p - value and log2 - FC . ( Black = Log2 - FC ≤ 1 & adj . p - value ≥ 0 . 05 , orange = Log2 - FC > 1 & adj . p - value ≥ 0 . 05 , red = Log2 - FC > 1 & adj . p - value < 0 . 05 ) . FC = fold change . B . STAT3 immunohistochemistry - staining of wild type , Ptenpc - / - , PtenStat3pc - / - mouse prostates . Scale bar = 100µm . E . Metabolite concentrations in nmol / µg of 5 metabolites in WT , Ptenpc - / - and PtenStat3pc - / - prostates . Box - plot shows median , 1st and 3rd quartiles , and whiskers extend to ± 1 . 5 interquartile range . Jitter represents biological replicates . ANOVA tests and Tukey multiple comparisons were applied . Red = PtenStat3pc - / - ( n = 3 ) , blue = Ptenpc - / - ( n = 5 ) , grey = wild type ( WT , n = 5 ) , q = adj . p . - value , n . s . = not significant .

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Figure 4A. Differentially expressed proteins in PtenStat3pc-/- vs. Ptenpc-/- proteomic samples. Colors indicate adj.p-value and log2-FC. (Black = Log2-FC ≤ 1 & adj. p-value ≥ 0.05, orange = Log2-FC > 1 & adj. p-value ≥ 0.05, red = Log2-FC > 1 & adj. p-value < 0.05). FC = fold change.B. STAT3 immunohistochemistry- staining of wild type, Ptenpc-/-, PtenStat3pc-/- mouse prostates. Scale bar = 100µm.E. Metabolite concentrations in nmol/µg of 5 metabolites in WT, Ptenpc-/- and PtenStat3pc-/- prostates. Box-plot shows median, 1st and 3rd quartiles, and whiskers extend to ± 1.5 interquartile range. Jitter represents biological replicates. ANOVA tests and Tukey multiple comparisons were applied. Red = PtenStat3pc-/- (n = 3), blue = Ptenpc-/- (n = 5), grey = wild type (WT, n=5), q = adj. p.-value, n.s. = not significant.


Figure 5A . SDHB protein expression levels in a TMA ( n = 83 ) detected by immunohistochemistry ( IHC ) . Box - plot shows median , 1st and 3rd quartiles , and whiskers extend to ± 1 . 5 interquartile range . Jitter represents single values in groups . Kruskal - Wallis test and Dunn ' s all pairs test were applied . Q = adj . p . - value , n . s . = not significant . TMA = Tissue Micro Array . B . Representative IHC - staining of SDHB in normal prostate glands and Gleason grade ( GL ) 3 - 5 PCa glands . Scale bar = 100µm . C . IDH2 protein expression levels in a TMA ( n = 83 ) detected by IHC . Box - plot shows median , 1st and 3rd quartiles , and whiskers extend to ± 1 . 5 interquartile range . Jitter represents single values in groups . Kruskal - Wallis test and Dunn ' s all pairs test were applied . Q = adj . p . - value , n . s . = not significant . D . Representative IHC staining of IDH2 in normal prostate glands and GL 3 - 5 PCa glands . Scale bar = 100µm . E . Nuclear ( N ) and cytoplasmatic ( C ) STAT3 protein expression levels in a TMA ( n = 83 ) detected by IHC . Box - plot shows median , 1st and 3rd quartiles , and whiskers extend to ± 1 . 5 interquartile range . Jitter represents single values in groups . Kruskal - Wallis test and Dunn ' s all pairs test were applied . Q = adj . p . - value , n . s . = not significant .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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

Figure 5A. SDHB protein expression levels in a TMA (n = 83) detected by immunohistochemistry (IHC). Box-plot shows median, 1st and 3rd quartiles, and whiskers extend to ± 1.5 interquartile range. Jitter represents single values in groups. Kruskal-Wallis test and Dunn's all pairs test were applied. Q = adj. p.-value, n.s. = not significant. TMA = Tissue Micro Array.B. Representative IHC- staining of SDHB in normal prostate glands and Gleason grade (GL) 3 - 5 PCa glands. Scale bar = 100µm.C. IDH2 protein expression levels in a TMA (n = 83) detected by IHC. Box-plot shows median, 1st and 3rd quartiles, and whiskers extend to ± 1.5 interquartile range. Jitter represents single values in groups. Kruskal-Wallis test and Dunn's all pairs test were applied. Q = adj. p.-value, n.s. = not significant.D. Representative IHC staining of IDH2 in normal prostate glands and GL 3 - 5 PCa glands. Scale bar = 100µm.E. Nuclear (N) and cytoplasmatic (C) STAT3 protein expression levels in a TMA (n = 83) detected by IHC. Box-plot shows median, 1st and 3rd quartiles, and whiskers extend to ± 1.5 interquartile range. Jitter represents single values in groups. Kruskal-Wallis test and Dunn's all pairs test were applied. Q = adj. p.-value, n.s. = not significant.


Figure 6B . Genes of the Hallmark gene set " Oxidative phosphorylation " that are differentially expressed in low STAT3 vs . high STAT3 tumors . Dotted lines show Log2 - FC = ± 1 ( x - axis ) and adj . p - value = - log10 ( 0 . 05 ) ( y - axis ) . Orange = up - regulated genes , green = down - regulated genes . DE = differentially expressed . C . - D . Kaplan - Meier plots showing time to biochemical recurrence in months for PDK4 in primary tumors ( C ) and in primary and metastatic tumors combined ( D ) in the MSKCC PCa GSE21032 data set . Groups were generated by a median split . P - values were estimated by Log - rank test ( C - D ) and adjusted with Benjamini - Hochberg method ( D ) . + = censored .

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 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

Figure 6B. Genes of the Hallmark gene set "Oxidative phosphorylation" that are differentially expressed in low STAT3 vs. high STAT3 tumors. Dotted lines show Log2-FC = ±1 (x-axis) and adj. p-value = -log10(0.05) (y-axis). Orange = up-regulated genes, green = down-regulated genes. DE = differentially expressed.C.- D. Kaplan-Meier plots showing time to biochemical recurrence in months for PDK4 in primary tumors (C) and in primary and metastatic tumors combined (D) in the MSKCC PCa GSE21032 data set. Groups were generated by a median split. P-values were estimated by Log-rank test (C-D) and adjusted with Benjamini-Hochberg method (D). + = censored.


Figure 7A . STAT3 and PDK4 stratified subgroups were generated by median splits in MSKCC PCa GSE21032 data set . Pearson correlation between STAT3 and PDK4 is shown . Kaplan - Meier plot shows stratified subgroups . P - values were estimated by Log - rank test and adjusted with Benjamini - Hochberg method . Hi = high , lo = low . B . Western blot of STAT3 , PDK4 and β - TUBULIN proteins in 22Rv1 cells with or without knockdown of STAT3 . Ctrl = scrambled control , shSTAT3 = short hairpin knockdown of STAT3 . C . ChIP assay from IL - 6 stimulated or non - stimulated 22Rv1 cells with or without knockdown of STAT3 was immunoprecipitated with a STAT3 specific antibody ( blue shades ) and IgG antibody as negative control ( orange shades ) followed by qPCR with a promoter - specific primer pair for the PDK4 gene . Bars represent mean ± SD from 2 technical replicates . Precipitated DNA is presented as % of input . One representative experiment is shown . Result of qPCR using primer pair 2 is shown . Ctrl = scrambled control , shSTAT3 = short hairpin knockdown of STAT3 , + IL - 6 = IL - 6 stimulated . D . Correlation of STAT3 , c - MYC and HIF - 1α with PDK1 - 4 , PDC - genes ( PDHA1 , PDHB , PDHX , DLAT , DLD ) and TCA / OXPHOS genes ( CS , IDH2 , IDH3A , SDHB , SDHC , ATP5A1 , NDUFS1 ) in MSKCC PCa ( GSE21032 ) . Dot colors represent Pearson correlation ( 1 = red , - 1 = blue ) , dot sizes represent adj . p - values ≤ 0 . 05 . Only significant correlations are shown . P - values were adjusted with Benjamini - Hochberg method .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 7A. STAT3 and PDK4 stratified subgroups were generated by median splits in MSKCC PCa GSE21032 data set. Pearson correlation between STAT3 and PDK4 is shown. Kaplan-Meier plot shows stratified subgroups. P-values were estimated by Log-rank test and adjusted with Benjamini-Hochberg method. Hi = high, lo = low.B. Western blot of STAT3, PDK4 and β-TUBULIN proteins in 22Rv1 cells with or without knockdown of STAT3. Ctrl = scrambled control, shSTAT3 = short hairpin knockdown of STAT3.C. ChIP assay from IL-6 stimulated or non-stimulated 22Rv1 cells with or without knockdown of STAT3 was immunoprecipitated with a STAT3 specific antibody (blue shades) and IgG antibody as negative control (orange shades) followed by qPCR with a promoter- specific primer pair for the PDK4 gene. Bars represent mean ± SD from 2 technical replicates. Precipitated DNA is presented as % of input. One representative experiment is shown. Result of qPCR using primer pair 2 is shown. Ctrl = scrambled control, shSTAT3 = short hairpin knockdown of STAT3, +IL-6 = IL-6 stimulated.D. Correlation of STAT3, c-MYC and HIF-1α with PDK1-4, PDC- genes (PDHA1, PDHB, PDHX, DLAT, DLD) and TCA/OXPHOS genes (CS, IDH2, IDH3A, SDHB, SDHC, ATP5A1, NDUFS1) in MSKCC PCa (GSE21032). Dot colors represent Pearson correlation (1 = red, -1 = blue), dot sizes represent adj. p-values ≤ 0.05. Only significant correlations are shown. P-values were adjusted with Benjamini-Hochberg method.


Figure 1 ( A ) BMDMs were treated with E . coli ( MOI = 100 ) for 30 minutes and further cultured for various length of time as indicated . The dynamic of ENT3 expression was determined by measuring Slc29a3 transcripts via RT - qPCR . ( B ) Phagocytosis inhibitor , Cytochalasin D ( CytoD ) , pretreated BMDMs were incubated with E . coli ( MOI = 100 ) and subjected for Slc29a3 expression analysis via RT - qPCR 6 hours post - infection . Data information : The expression level was calculated relative to Rpl19 and normalized to untreated group as 1 . The results shown are combined from three biological replicates ( n = 3 ) , unpaired two - tailed Student ' s t - test was used for statistical analysis , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . no significance . Data are shown as mean ± SEM . ( D ) Different bacterial components were applied as stimulants to BMDMs . Slc29a3 expression was analyzed after 30 minutes of stimulation and a total of 6 hours incubation . ( E ) BMDMs were treated with different doses of LPS for 30 minutes , harvested 6 hours post - stimulation , and subjected for RT - qPCR analysis . Data information : The expression level was calculated relative to Rpl19 and normalized to untreated group as 1 . The results shown are combined from three biological replicates ( n = 3 ) , unpaired two - tailed Student ' s t - test was used for statistical analysis , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . no significance . Data are shown as mean ± SEM .

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 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

Figure 1(A) BMDMs were treated with E. coli (MOI=100) for 30 minutes and further cultured for various length of time as indicated. The dynamic of ENT3 expression was determined by measuring Slc29a3 transcripts via RT-qPCR. (B) Phagocytosis inhibitor, Cytochalasin D (CytoD), pretreated BMDMs were incubated with E. coli (MOI=100) and subjected for Slc29a3 expression analysis via RT-qPCR 6 hours post-infection. Data information: The expression level was calculated relative to Rpl19 and normalized to untreated group as 1. The results shown are combined from three biological replicates (n=3), unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.(D) Different bacterial components were applied as stimulants to BMDMs. Slc29a3 expression was analyzed after 30 minutes of stimulation and a total of 6 hours incubation. (E) BMDMs were treated with different doses of LPS for 30 minutes, harvested 6 hours post-stimulation, and subjected for RT-qPCR analysis. Data information: The expression level was calculated relative to Rpl19 and normalized to untreated group as 1. The results shown are combined from three biological replicates (n=3), unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.


Figure 2 ( C ) WT or ( D ) MyD88 - / - BMDMs were stimulated with 10 ng / ml LPS or live E . coli ( MOI = 100 ) in the presence of 10 µM TBK inhibitor , MRT 67307 , and analyzed for Slc29a3 expression . Data information : The results shown are combined from three biological replicates ( n = 3 ) , unpaired two - tailed Student ' s t - test was used for statistical analysis , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . no significance . Data are shown as mean ± SEM . ( E ) The 10 ng / ml LPS - treated BMDMs were examined for Ifnb1 expression after 30 minutes of stimulation and a total of 6 hours incubation . Data information : The results shown are combined from three biological replicates ( n = 3 ) , unpaired two - tailed Student ' s t - test was used for statistical analysis , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . no significance . Data are shown as mean ± SEM . ( F ) BMDMs were treated with 100 U / ml of IFN - β for various length of time and subjected for Slc29a3 expression . Data information : The results shown are combined from three biological replicates ( n = 3 ) , unpaired two - tailed Student ' s t - test was used for statistical analysis , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . no significance . Data are shown as mean ± SEM .

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 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

Figure 2(C) WT or (D) MyD88-/- BMDMs were stimulated with 10 ng/ml LPS or live E. coli (MOI=100) in the presence of 10 µM TBK inhibitor, MRT 67307, and analyzed for Slc29a3 expression. Data information: The results shown are combined from three biological replicates (n=3), unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.(E) The 10 ng/ml LPS-treated BMDMs were examined for Ifnb1 expression after 30 minutes of stimulation and a total of 6 hours incubation. Data information: The results shown are combined from three biological replicates (n=3), unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.(F) BMDMs were treated with 100 U/ml of IFN-β for various length of time and subjected for Slc29a3 expression. Data information: The results shown are combined from three biological replicates (n=3), unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.


Figure 3 ( B - E ) BMDMs from WT , IFNAR1 - / - ( B ) , or STAT1 - / - ( C ) mice were treated with 10 ng / ml LPS , live E . coli ( MOI = 100 ) , or 100 U / ml IFN - β , harvested and examined for Slc29a3 induction . BMDMs from WT , IFNAR1 - / - ( D ) or STAT1 - / - ( E ) mice were treated with 100 U / ml IFN - β , 50 µg / ml poly ( I : C ) , or EMCV ( MOI = 10 ) , harvested and examined for Slc29a3 induction . Data information : The expression level was calculated relative to Rpl19 and normalized to untreated group as 1 . The results shown in B to E are from three biological replicates ( n = 3 ) Unpaired two - tailed Student ' s t - test was used for statistical analysis , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . no significance . Data are shown as mean ± SEM . ( F - H ) The expression dynamic of Slc29a3 was profiled in BMDMs treated with two different doses of IFN - α ( F ) , IFN - β ( G ) , IFN - γ ( H ) for various length of time as indicated . Data information : The expression level was calculated relative to Rpl19 and normalized to untreated group as 1 . The results shown in F to H from two biological replicates ( n = 2 ) . Unpaired two - tailed Student ' s t - test was used for statistical analysis , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . no significance . Data are shown as mean ± SEM .

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 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

Figure 3(B-E) BMDMs from WT, IFNAR1-/- (B), or STAT1-/- (C) mice were treated with 10 ng/ml LPS, live E. coli (MOI=100), or 100 U/ml IFN-β, harvested and examined for Slc29a3 induction. BMDMs from WT, IFNAR1-/- (D) or STAT1-/- (E) mice were treated with 100 U/ml IFN-β, 50 µg/ml poly(I:C), or EMCV (MOI=10), harvested and examined for Slc29a3 induction. Data information: The expression level was calculated relative to Rpl19 and normalized to untreated group as 1. The results shown in B to E are from three biological replicates (n=3) Unpaired two-tailed Student's t-test was used for statistical analysis, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.(F-H) The expression dynamic of Slc29a3 was profiled in BMDMs treated with two different doses of IFN-α (F), IFN-β (G), IFN-γ (H) for various length of time as indicated. Data information: The expression level was calculated relative to Rpl19 and normalized to untreated group as 1. The results shown in F to H from two biological replicates (n=2). Unpaired two-tailed Student's t-test was used for statistical analysis, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.


Figure 4 ( A ) The induction fold change ( treated / untreated ) of Slc29a3 and selected ISG transcripts were curated from INTERFEROME database . Shown is data extracted from 250 IU / ml IFN - β treated BMDMs . Data information : Unpaired two - tailed Student ' s t - test was used for statistical analysis , * * * p < 0 . 001 , n . s . no significance . Data are shown as mean ± SEM . ( D - F ) Primers targeting 3 ' flanking region 2 ( D ) , 3 ' flanking region 1 ( E ) , and promoter region ( F ) were used to quantify the STAT1 binding in BMDMs upon 100 U / ml IFN - β after 4 hour stimulation via qChIP assay . Data information : The results in D - F were generated from two biological replicates ( n = 2 ) with four technical repeats each ( n = 4 ) . One representative example is shown . Unpaired two - tailed Student ' s t - test was used for statistical analysis , * * * p < 0 . 001 , n . s . no significance . Data are shown as mean ± SEM .

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 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

Figure 4(A) The induction fold change (treated/untreated) of Slc29a3 and selected ISG transcripts were curated from INTERFEROME database. Shown is data extracted from 250 IU/ml IFN-β treated BMDMs. Data information: Unpaired two-tailed Student's t-test was used for statistical analysis, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.(D-F) Primers targeting 3' flanking region 2 (D), 3' flanking region 1 (E), and promoter region (F) were used to quantify the STAT1 binding in BMDMs upon 100 U/ml IFN-β after 4 hour stimulation via qChIP assay. Data information: The results in D-F were generated from two biological replicates (n=2) with four technical repeats each (n=4). One representative example is shown. Unpaired two-tailed Student's t-test was used for statistical analysis, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.


Figure 5 ( A - C ) ( A ) BMDMs were infected with EMCV ( MOI = 10 ) and harvested at indicated time points . ( B ) THP - 1 derived macrophages were challenged with EV71 ( MOI = 5 ) and collected at different time points . ( C ) BMDMs were co - cultured with 50 µg / ml poly ( I : C ) with various length of time and harvested . The mRNA expression of Slc29a3 or SLC29A3 was measured by RT - qPCR . The expression level was calculated relative to Rpl19 or RPLP0 and normalized to untreated group at time 0 as 1 . Results are combined from three biological replicates ( n = 3 ) Data information : Unpaired two - tailed Student ' s t - test was used for statistical analysis , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . no significance . Data are shown as mean ± SEM . ( D , E ) ENT3fl / fl and Cx3cr1cre / + ENT3fl / fl mice ( n = 12 ) were challenged with EMCV ( 104 PFU , i . p . ) and monitored for the survival ( D ) and disease severity ( E ) for two weeks . Survival results were analyzed by log - rank ( Mantel - Cox ) test , * p < 0 . 05 . Data information : Unpaired two - tailed Student ' s t - test was used for statistical analysis , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . no significance . Data are shown as mean ± SEM . ( F , G ) ENT3fl / fl and Cx3cr1cre / + ENT3fl / fl mice were challenged with 107 PFU EMCV and measured the viral titer in the brain at post - infection day 3 by plaque assay ( n = 6 ) . Shown are representative plaque assay images ( F ) and the quantification ( G ) . Data information : Unpaired two - tailed Student ' s t - test was used for statistical analysis , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . no significance . Data are shown as mean ± SEM .

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 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 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

Figure 5(A-C) (A) BMDMs were infected with EMCV (MOI=10) and harvested at indicated time points. (B) THP-1 derived macrophages were challenged with EV71 (MOI=5) and collected at different time points. (C) BMDMs were co-cultured with 50 µg/ml poly (I:C) with various length of time and harvested. The mRNA expression of Slc29a3 or SLC29A3 was measured by RT-qPCR. The expression level was calculated relative to Rpl19 or RPLP0 and normalized to untreated group at time 0 as 1. Results are combined from three biological replicates (n=3) Data information: Unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.(D, E) ENT3fl/fl and Cx3cr1cre/+ENT3fl/fl mice (n=12) were challenged with EMCV (104 PFU, i.p.) and monitored for the survival (D) and disease severity (E) for two weeks. Survival results were analyzed by log-rank (Mantel-Cox) test, *p < 0.05. Data information: Unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.(F, G) ENT3fl/fl and Cx3cr1cre/+ENT3fl/fl mice were challenged with 107 PFU EMCV and measured the viral titer in the brain at post-infection day 3 by plaque assay (n=6). Shown are representative plaque assay images (F) and the quantification (G). Data information: Unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.


Figure 6 ( A ) WT and ENT3 - / - BMDMs were infected with EMCV ( MOI = 10 ) , and the culture supernatants were harvested 24 hours post - infection to measure the production of IFN - α , IFN - β , and IFN - γ by ELISA . Shown are combined results from three biological replicates ( n = 3 ) . Data information : Unpaired two - tailed Student ' s t - test was used for statistical analysis , * p < 0 . 05 , * * * p < 0 . 001 . n . s . no significance . The combined data are shown as mean ± SEM . ( D , E ) The mitochondrial respiratory capacity ( D ) and glycolysis capacity ( E ) of WT and ENT3 - / - BMDMs treated with 1000 U / ml IFN - β for 6 hours were measured by extracellular flux analysis . Shown are the representative results of OCR and ECAR kinetics from six biological replicates ( n = 6 ) . Data information : Unpaired two - tailed Student ' s t - test was used for statistical analysis , * p < 0 . 05 , * * * p < 0 . 001 . n . s . no significance . The combined data are shown as mean ± SEM . ( G , H ) The replication of EMCV in WT ( G ) and ENT3 - / - BMDMs ( H ) were evaluated in the presence of exogenous 1 or 10 µM of ribonucleosides ( A , U , C , G ) 9 hours post - infection ( MOI = 10 ) . The expression of EMCV - 2A2B was determined by RT - qPCR . Shown are results from three to four biological replicates ( n = 3 - 4 ) . Data information : In G , H ) the EMCV - 2A2B expression level was calculated relative to Rpl19 and normalized to untreated group at time 0 as 1 . Unpaired two - tailed Student ' s t - test was used for statistical analysis , * p < 0 . 05 , * * * p < 0 . 001 . n . s . no significance . The combined data are shown as mean ± SEM .

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 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 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

Figure 6(A) WT and ENT3-/- BMDMs were infected with EMCV (MOI=10), and the culture supernatants were harvested 24 hours post-infection to measure the production of IFN-α, IFN-β, and IFN-γ by ELISA. Shown are combined results from three biological replicates (n=3). Data information: Unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, ***p < 0.001. n.s. no significance. The combined data are shown as mean ± SEM.(D, E) The mitochondrial respiratory capacity (D) and glycolysis capacity (E) of WT and ENT3-/- BMDMs treated with 1000 U/ml IFN-β for 6 hours were measured by extracellular flux analysis. Shown are the representative results of OCR and ECAR kinetics from six biological replicates (n=6). Data information: Unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, ***p < 0.001. n.s. no significance. The combined data are shown as mean ± SEM.(G, H) The replication of EMCV in WT (G) and ENT3-/- BMDMs (H) were evaluated in the presence of exogenous 1 or 10 µM of ribonucleosides (A, U, C, G) 9 hours post-infection (MOI=10). The expression of EMCV-2A2B was determined by RT-qPCR. Shown are results from three to four biological replicates (n=3-4). Data information: In G, H) the EMCV-2A2B expression level was calculated relative to Rpl19 and normalized to untreated group at time 0 as 1. Unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, ***p < 0.001. n.s. no significance. The combined data are shown as mean ± SEM.


Figure 7 ( A ) Illustration of strategy to track the viral uncoating upon infection . ( B - D ) ( B ) WT and ENT3 - / - BMDMs were infected with Syto82 - labeled EMCV ( MOI = 1 ) and subjected to live imagining 30 , 60 and 90 minutes post - infection . The arrowheads indicated the Syto82 + virus puncta in the cell . Scale bars represent 10 µm . ( C ) Quantification of Syto82 signal in BMDMs after 60 minutes was performed by enumerating Syto82 + cells ( % ) and ( D ) the mean fluorescent intensity ( MFI ) per Syto82 + cell ( D ) . Results were generated from images of three biological replicates ( n = 3 ) , each with 4 fields analyzed . Data information : Unpaired two - tailed Student ' s t - test was used for statistical analysis , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . no significance . Data are shown as mean ± SEM . ( E ) WT and ENT3 - / - BMDMs were infected with Syto82 - labeled EMCV ( MOI = 1 ) and co - stained with 100 nM Lysotracker Green , subjected to live imagining 60 minutes post - infection . Data were the representative images of two biological replicates ( n = 2 ) . Scale bars represent 5 µm . ( F ) WT and ENT3 - / - BMDMs were infected with EMCV ( MOI = 10 ) for 2 hours and then enriched the endosomes and lysosomes . EMCV particles in endosome - or lysosome - enriched fractions were quantified by plaque assay . Shown are combined results from three biological replicates ( n = 3 ) . Data information : Unpaired two - tailed Student ' s t - test was used for statistical analysis , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . no significance . Data are shown as mean ± SEM . ( G - K ) ( G ) Calu - 3 cells were infected with SARS - CoV - 2 original strain or delta variant at MOI = 0 . 1 for 48 hours , harvested , and examined for SLC29A3 induction . ( H ) ENT3 knockdown in Calu - 3 cells was performed by lentiviral shRNA system . The knockdown efficiency was confirmed by RT - qPCR . ENT3 knockdown Calu - 3 cells were infected with SARS - CoV - 2 delta variant and subjected for the viral replication analysis 24 hours post - infection . The virus released out of cells was measured by plaque assay ( I ) , and the viral titer in the cells was measured by RT - qPCR ( J ) . ( K ) The replication of SARS - CoV - 2 delta variant in scramble and ENT3 knockdown Calu - 3 cells were evaluated in the presence of exogenous 1 or 10 µM of ribonucleosides ( A , U , C , G ) 24 hours post - infection ( MOI = 0 . 1 ) . The SLC29A3 expression level was calculated relative to RPLP0 and normalized to control as 1 . The SARS - CoV - 2 E gene expression level was calculated relative to RPLP0 to normalize the cell number . Shown are combined results from three biological replicates ( n = 3 ) . Data information : Unpaired two - tailed Student ' s t - test was used for statistical analysis , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . no significance . Data are shown as mean ± SEM .

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 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 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 O O O O O O

Figure 7(A) Illustration of strategy to track the viral uncoating upon infection. (B-D) (B) WT and ENT3-/- BMDMs were infected with Syto82-labeled EMCV (MOI=1) and subjected to live imagining 30, 60 and 90 minutes post-infection. The arrowheads indicated the Syto82+ virus puncta in the cell. Scale bars represent 10 µm. (C) Quantification of Syto82 signal in BMDMs after 60 minutes was performed by enumerating Syto82+ cells (%) and (D) the mean fluorescent intensity (MFI) per Syto82+ cell (D). Results were generated from images of three biological replicates (n=3), each with 4 fields analyzed. Data information: Unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.(E) WT and ENT3-/- BMDMs were infected with Syto82-labeled EMCV (MOI=1) and co-stained with 100 nM Lysotracker Green, subjected to live imagining 60 minutes post-infection. Data were the representative images of two biological replicates (n=2). Scale bars represent 5 µm. (F) WT and ENT3-/- BMDMs were infected with EMCV (MOI=10) for 2 hours and then enriched the endosomes and lysosomes. EMCV particles in endosome- or lysosome-enriched fractions were quantified by plaque assay. Shown are combined results from three biological replicates (n=3). Data information: Unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.(G-K) (G) Calu-3 cells were infected with SARS-CoV-2 original strain or delta variant at MOI=0.1 for 48 hours, harvested, and examined for SLC29A3 induction. (H) ENT3 knockdown in Calu-3 cells was performed by lentiviral shRNA system. The knockdown efficiency was confirmed by RT-qPCR. ENT3 knockdown Calu-3 cells were infected with SARS-CoV-2 delta variant and subjected for the viral replication analysis 24 hours post-infection. The virus released out of cells was measured by plaque assay (I), and the viral titer in the cells was measured by RT-qPCR (J). (K) The replication of SARS-CoV-2 delta variant in scramble and ENT3 knockdown Calu-3 cells were evaluated in the presence of exogenous 1 or 10 µM of ribonucleosides (A, U, C, G) 24 hours post-infection (MOI=0.1). The SLC29A3 expression level was calculated relative to RPLP0 and normalized to control as 1. The SARS-CoV-2 E gene expression level was calculated relative to RPLP0 to normalize the cell number. Shown are combined results from three biological replicates (n=3). Data information: Unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.


Figure 1Western blotting showing the expression of RAP1 and TRF2 in MRC - 5 cells of different population doublings ( PD ) . Senescent cells ( PD 72 ) were left in culture for at least 4 weeks before harvesting for analysis . ChIP analysis of young ( PD 30 ) and senescent ( PD 72 ) MRC - 5 cells performed with either anti - RAP1 , anti - TRF2 or an IgG antibody . The immunoprecipitated and input products were loaded into a slot blot membrane and hybridized with a telomere probe , stripped and hybridized to an Alu probe in order to determine unspecific binding . Quantification was performed by normalizing the telomere signal of the immunoprecipitated to that of the input . Data represent mean + / - SD of three biological replicates . Immunofluorescence detection of 53BP1 ( red ) and a FISH probe staining telomeres ( green ) in young ( PD 26 ) , pre - senescent ( PD 66 ) and senescent ( PD 72 + 4 weeks ) MRC - 5 fibroblasts . The upper graph shows the percentage of telomere co - localizing with 53BP1 ( TIFs ) . Total DNA damage was measured by immunofluourescence detection of 53BP1 . The lower graph shows the total number of 53BP1 foci per nucleus . Approximately 40 - 50 cells were analyzed per replicate and per condition . Bars represent SEM of 2 biological replicates .

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 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 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

Figure 1Western blotting showing the expression of RAP1 and TRF2 in MRC-5 cells of different population doublings (PD). Senescent cells (PD 72) were left in culture for at least 4 weeks before harvesting for analysis.ChIP analysis of young (PD 30) and senescent (PD 72) MRC-5 cells performed with either anti-RAP1, anti-TRF2 or an IgG antibody. The immunoprecipitated and input products were loaded into a slot blot membrane and hybridized with a telomere probe, stripped and hybridized to an Alu probe in order to determine unspecific binding. Quantification was performed by normalizing the telomere signal of the immunoprecipitated to that of the input. Data represent mean +/- SD of three biological replicates.Immunofluorescence detection of 53BP1 (red) and a FISH probe staining telomeres (green) in young (PD 26), pre-senescent (PD 66) and senescent (PD 72 + 4 weeks) MRC-5 fibroblasts. The upper graph shows the percentage of telomere co-localizing with 53BP1 (TIFs). Total DNA damage was measured by immunofluourescence detection of 53BP1. The lower graph shows the total number of 53BP1 foci per nucleus. Approximately 40-50 cells were analyzed per replicate and per condition. Bars represent SEM of 2 biological replicates.


Figure 2Number of telomere fusions performed by PCR using 3 subtelomeric primers in the same reaction ( 16p1 , 21q1 and XpYpM ) . The primer 16p1 is able to bind the subtelomeric region of 1p 9q , 12p , 15q , 16p , XqYq and the interstitial 2q14 region ; the 21q1 primer binds the subtelomeric region of 1q , 2q , 5q , 6q , 6p , 8p , 10q , 13q , 19p , 19q , 21q , 22q and the interstitial 2q13 site . The PCR - fusion assay was performed in MRC - 5 primary fibroblasts of different population doublings ( PD ) with or without depletion of RAP1 ( shRAP1 or shControl respectively ) . Lentiviral infection was performed for 10 days . Data represent mean + / - SD of 3 biological replicates . Statistical analyses were performed using Mann - Whitney U test ( * * p < 0 . 001 ; * * * p < 0 . 0001 ) . Southern blotting showing the hybridization of the 16p probe for the conditions described in A . Number of fusions per 1 µg of DNA is shown at the bottom of the gels . Number of fusions per 1 µg of DNA in MRC - 5 senescent cells . Lentiviral transfections with shRNAs were carried out for 10 days while transfections with siRNAs for 6 days ( two subsequent transfections of 3 days each ) . Representative southern blot membranes hybridized with the 16p probe are shown . Data represent mean + / - SD of 3 biological replicates . Statistical analyses were performed using Mann - Whitney U test ( * p < 0 . 05 ; * * p < 0 . 001 ; * * * p < 0 . 0001 ) .

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 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

Figure 2Number of telomere fusions performed by PCR using 3 subtelomeric primers in the same reaction (16p1, 21q1 and XpYpM). The primer 16p1 is able to bind the subtelomeric region of 1p 9q, 12p, 15q, 16p, XqYq and the interstitial 2q14 region; the 21q1 primer binds the subtelomeric region of 1q, 2q, 5q, 6q, 6p, 8p, 10q, 13q, 19p, 19q, 21q, 22q and the interstitial 2q13 site. The PCR-fusion assay was performed in MRC-5 primary fibroblasts of different population doublings (PD) with or without depletion of RAP1 (shRAP1 or shControl respectively). Lentiviral infection was performed for 10 days. Data represent mean +/- SD of 3 biological replicates. Statistical analyses were performed using Mann-Whitney U test (**p < 0.001; ***p < 0.0001). Southern blotting showing the hybridization of the 16p probe for the conditions described in A. Number of fusions per 1 µg of DNA is shown at the bottom of the gels.Number of fusions per 1 µg of DNA in MRC-5 senescent cells. Lentiviral transfections with shRNAs were carried out for 10 days while transfections with siRNAs for 6 days (two subsequent transfections of 3 days each). Representative southern blot membranes hybridized with the 16p probe are shown. Data represent mean +/- SD of 3 biological replicates. Statistical analyses were performed using Mann-Whitney U test (*p < 0.05; **p < 0.001; ***p < 0.0001).


Figure 3Distribution of the telomere repeat array found at the junction between two fused chromosomes of senescent cells transduced with lentiviral vectors expressing either shControl or shRAP1 . Variant telomere repeats were included in the estimation of the telomere array length . Data represent median + / - interquartile range ( green lines ) of 3 biological replicates . Statistical analysis was performed using Mann - Whitney U test * * * p < 0 . 0001 ) .

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

Figure 3Distribution of the telomere repeat array found at the junction between two fused chromosomes of senescent cells transduced with lentiviral vectors expressing either shControl or shRAP1. Variant telomere repeats were included in the estimation of the telomere array length. Data represent median +/- interquartile range (green lines) of 3 biological replicates. Statistical analysis was performed using Mann-Whitney U test ***p < 0.0001).


Figure 4RAP1 expression in HeLa cells treated with the telomerase inhibitor BIBR1532 for 25 days ( 20 µM final concentration ) . Fifteen days before cell harvesting , doxycycline ( DOX ; 1 µg / µl final concentration ) was added to deplete the expression of RAP1 . Telomere length analysis by southern blotting of the samples described in A . The size of the main intensity peak is indicated at the bottom of the gel . Number of fusions in HeLa cells after 25 days in culture . Cells were maintained with BIBR1532 during the whole period of the experiment , while doxycycline ( 1 µg / µl final concentration ) and the infection with shControl , shLIG3 or shLIG4 was carried out for the last 15 days of the experiment . Data represent mean + / - SD of three biological replicates are shown . Statistical analyses were performed using Mann - Whitney U test ( * p < 0 . 05 ; * * p < 0 . 001 ; * * * p < 0 . 0001 ) . Examples of metaphase spreads hybridized with a telomeric PNA probe ( green ) when RAP1 was depleted ( + DOX ) . Telomerase was inhibited with BIBR1532 for 25 days . Scale bar = 10 µm . Quantification of chromosome aberrations observed in RAP1 depleted cells ( + DOX ) or control ( - DOX ) . Data represent mean + / - SD of three biological replicates . Approximately 15 metaphase spreads were analyzed per replicate with a total of 2300 chromosomes examined per condition . * p < 0 . 05 ; two - tailed Student ' s t test .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 4RAP1 expression in HeLa cells treated with the telomerase inhibitor BIBR1532 for 25 days (20 µM final concentration). Fifteen days before cell harvesting, doxycycline (DOX; 1 µg/µl final concentration) was added to deplete the expression of RAP1.Telomere length analysis by southern blotting of the samples described in A. The size of the main intensity peak is indicated at the bottom of the gel.Number of fusions in HeLa cells after 25 days in culture. Cells were maintained with BIBR1532 during the whole period of the experiment, while doxycycline (1 µg/µl final concentration) and the infection with shControl, shLIG3 or shLIG4 was carried out for the last 15 days of the experiment. Data represent mean +/- SD of three biological replicates are shown. Statistical analyses were performed using Mann-Whitney U test (*p < 0.05; **p < 0.001; ***p < 0.0001).Examples of metaphase spreads hybridized with a telomeric PNA probe (green) when RAP1 was depleted (+DOX). Telomerase was inhibited with BIBR1532 for 25 days. Scale bar = 10 µm. Quantification of chromosome aberrations observed in RAP1 depleted cells (+DOX) or control (-DOX). Data represent mean +/- SD of three biological replicates. Approximately 15 metaphase spreads were analyzed per replicate with a total of 2300 chromosomes examined per condition. *p < 0.05; two-tailed Student's t test.


Figure 5Growth curve of MRC - 5 fibroblasts . Cells were grown until senescence and after three weeks of no growth detection , lentiviral infections containing either shp21CIP1 or shp21CIP1 + shRAP1 sequences were performed . Cells were harvested 15 days post infection ( dpi ) . Data represent mean + / - SD of three biological replicates measured after 8 and 15 dpi . Expression of p21CIP1 and RAP1 after 15 dpi of cells transduced with shControl , shp21CIP1 or shp21CIP1 + shRAP1 . Dividing cells ( magenta ) were identified by EdU staining ( 1 µM EdU for 24 h ) . The percentage of dividing cells after 15 dpi is shown ( n = 3 ) . Scale bar = 10 µm . Senescence - associated β - galactosidase ( SA - β - gal ) staining in senescent and post - senescent fibroblasts ( 1 and 15 dpi ) is shown . Percentage of SA - β - gal positive cells is indicated for each condition . Three biological replicates were performed , with approximately 300 cells analyzed per condition . Scale bar = 50 µm . Senescent cells , transduced with shp21CIP1 or shp21CIP1 + shRAP1 for 15 days , were stained with CytoCalcein violet ( live cells ) , Apopxin Green ( apoptotic cells ) and 7 - aminoactinomycin D ( 7 - AAD ) ( late apoptostic / necrotic cells ) and visualized by flow cytometry . Quantification of apoptotic cells from the conditions described in E . Data represent mean + / - SD of three biological replicates ( * * P < 0 . 001 ; two - tailed Student ' s t test ) .

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Figure 5Growth curve of MRC-5 fibroblasts. Cells were grown until senescence and after three weeks of no growth detection, lentiviral infections containing either shp21CIP1 or shp21CIP1 + shRAP1 sequences were performed. Cells were harvested 15 days post infection (dpi). Data represent mean +/- SD of three biological replicates measured after 8 and 15 dpi.Expression of p21CIP1 and RAP1 after 15 dpi of cells transduced with shControl, shp21CIP1 or shp21CIP1 + shRAP1.Dividing cells (magenta) were identified by EdU staining (1 µM EdU for 24 h). The percentage of dividing cells after 15 dpi is shown (n = 3). Scale bar = 10 µm.Senescence-associated β-galactosidase (SA-β-gal) staining in senescent and post-senescent fibroblasts (1 and 15 dpi) is shown. Percentage of SA-β-gal positive cells is indicated for each condition. Three biological replicates were performed, with approximately 300 cells analyzed per condition. Scale bar = 50 µm.Senescent cells, transduced with shp21CIP1 or shp21CIP1+shRAP1 for 15 days, were stained with CytoCalcein violet (live cells), Apopxin Green (apoptotic cells) and 7-aminoactinomycin D (7-AAD) (late apoptostic/necrotic cells) and visualized by flow cytometry. Quantification of apoptotic cells from the conditions described in E. Data represent mean +/- SD of three biological replicates (**P < 0.001; two-tailed Student's t test).


Figure 1ATAC - seq peaks more accessible in GPR56low ( violet ) or GPR56high ( turquoise ) against the background ( grey ) are enriched for enhancers associated with specific hematopoietic cell types . P - values and odds ratios are given for a pairwise , two - sided Fisher ' s Exact test comparing each category ( GPR56low / high ) against the background . Enhancer annotations are taken from EnhancerAtlas 2 . 0 . * * * p < 0 . 0005 , * * p < 0 . 005 , * p < 0 . 05 . Knockdown efficiency of two shRNAs against GPR56 ( shGPR56weak and shGPR56strong ) versus shLuc as negative control measured on protein level by flow cytometry in CD34 + CB cells . Shown are representative FACS plots ( left ) and the percentage of GPR56 + cells of transduced GFP + cells on day 5 ( right panel ) . Biological N = 4 , unpaired t - test , bars and error bars represent mean and standard deviation . * * * p < 0 . 0005 , * * p < 0 . 005 , * p < 0 . 05 . Integrative Genome Viewer ( IGV ) snapshot showing ATAC - seq peaks along and upstream of the VWF gene in GPR56high vs . low samples ( top , average peak size of 10 GPR56high ( turquoise ) and 15 GPR56low samples ( violet ) ) , RNA - seq reads of the same location in AML samples with high ( n = 9 ) versus low ( n = 11 ) GPR56 expression ( two middle tracks ) , and RNA - seq reads of shLuc versus GPR56 knockdown CD34 + cells ( 3 bottom tracks , one of two replicates shown for each condition ) . Track height was group - scaled . Dashed vertical lines indicate binding sites for the annotated TFs . TFBS : transcription factor binding site derived from the HOCOMOCO v10 database ; TSS : transcription start site . TFs in blue bind to differentially accessible chromatin regions . Volcano plot of differential transcription factor ( TF ) motif accessibility ( activity ) in GPR56high ( turquoise ) vs GPR56low ( violet ) samples and their corresponding adjusted p - values determined with diffTF . Highlighted are TFs whose RNA expression was also positively or negatively affected by GPR56 KD in the RNA - seq dataset .

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Figure 1ATAC-seq peaks more accessible in GPR56low (violet) or GPR56high (turquoise) against the background (grey) are enriched for enhancers associated with specific hematopoietic cell types. P-values and odds ratios are given for a pairwise, two-sided Fisher's Exact test comparing each category (GPR56low / high) against the background. Enhancer annotations are taken from EnhancerAtlas 2.0. *** p<0.0005, ** p<0.005, * p<0.05.Knockdown efficiency of two shRNAs against GPR56 (shGPR56weak and shGPR56strong) versus shLuc as negative control measured on protein level by flow cytometry in CD34+ CB cells. Shown are representative FACS plots (left) and the percentage of GPR56+ cells of transduced GFP+ cells on day 5 (right panel). Biological N=4, unpaired t-test, bars and error bars represent mean and standard deviation. *** p<0.0005, ** p<0.005, * p<0.05.Integrative Genome Viewer (IGV) snapshot showing ATAC-seq peaks along and upstream of the VWF gene in GPR56high vs. low samples (top, average peak size of 10 GPR56high (turquoise) and 15 GPR56low samples (violet)), RNA-seq reads of the same location in AML samples with high (n=9) versus low (n=11) GPR56 expression (two middle tracks), and RNA-seq reads of shLuc versus GPR56 knockdown CD34+ cells (3 bottom tracks, one of two replicates shown for each condition). Track height was group-scaled. Dashed vertical lines indicate binding sites for the annotated TFs. TFBS: transcription factor binding site derived from the HOCOMOCO v10 database; TSS: transcription start site. TFs in blue bind to differentially accessible chromatin regions.Volcano plot of differential transcription factor (TF) motif accessibility (activity) in GPR56high (turquoise) vs GPR56low (violet) samples and their corresponding adjusted p-values determined with diffTF. Highlighted are TFs whose RNA expression was also positively or negatively affected by GPR56 KD in the RNA-seq dataset.


Figure 2Violin plots showing geometric mean ( horizontal line ) and individual values ( circles ) of the engraftment levels of different hematopoietic cell types in the bone marrow of NSG mice 20 weeks after transplantation of CB CD34 + cells following overnight - infection with shGPR56strong ( turqouise ) and shGPR56weak ( gray ) versus shLuc control ( violet ) . Shown are percentages of indicated populations , which are also co - positive for human CD45 and GFP , relative to all harvested bone marrow cells . HSPC : CD34 + SSClowCD33 - CD19 - , B precursor : CD34 + CD19 + CD33 - , myeloid progenitor ( myelo . prog . ) : CD34 + CD33 + CD19 - , mature B cells : CD19 + CD34 - , mature myeloid cells : CD33 + CD19 - CD34 - . Multiple unpaired t - tests of log - transformed values , p - values were Benjamini and Hochberg corrected ( padj ) , * padj < 0 . 05 , * * padj < 0 . 005 , * * * padj < 0 . 0005 . Adhesion of control and GPR56 KD K562 cells on fibronectin - functionalized substrates . Left : Overlay of phase contrast and microinterferometry images of control ( above ) and GPR56 KD cells ( below ) . The area of tight adhesion extracted by microinterferometry is highlighted in turquoise and violet , respectively . Right : Comparison of adhesion areas extracted by microinterferometry , reduction of adhesion area by factor 1 . 9 in GPR56 KD cells ( p = 1 . 0 x 10 - 10 , two - sided Mann - Whitney test , box plots showing medians , quartiles , and outliers according to the Tukey method ) . Technical replicates Ncontrol = 71 , Technical replicates NKD = 104 , scale bar 10 µm .

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Figure 2Violin plots showing geometric mean (horizontal line) and individual values (circles) of the engraftment levels of different hematopoietic cell types in the bone marrow of NSG mice 20 weeks after transplantation of CB CD34+ cells following overnight-infection with shGPR56strong (turqouise) and shGPR56weak (gray) versus shLuc control (violet). Shown are percentages of indicated populations, which are also co-positive for human CD45 and GFP, relative to all harvested bone marrow cells. HSPC: CD34+SSClowCD33-CD19-, B precursor: CD34+CD19+CD33-, myeloid progenitor (myelo. prog.): CD34+CD33+CD19-, mature B cells: CD19+CD34-, mature myeloid cells: CD33+CD19-CD34-. Multiple unpaired t-tests of log-transformed values, p-values were Benjamini and Hochberg corrected (padj), * padj<0.05, ** padj<0.005, *** padj<0.0005.Adhesion of control and GPR56 KD K562 cells on fibronectin-functionalized substrates. Left: Overlay of phase contrast and microinterferometry images of control (above) and GPR56 KD cells (below). The area of tight adhesion extracted by microinterferometry is highlighted in turquoise and violet, respectively. Right: Comparison of adhesion areas extracted by microinterferometry, reduction of adhesion area by factor 1.9 in GPR56 KD cells (p = 1.0 x 10-10, two-sided Mann-Whitney test, box plots showing medians, quartiles, and outliers according to the Tukey method). Technical replicates Ncontrol = 71, Technical replicates NKD = 104, scale bar 10 µm.


Figure 3 β - catenin protein expression in K562 cells infected with shGPR56strong or shLuc negative control in presence and absence of Wnt3a ( representative Western Blot ( above ) and quantification after normalization to GAPDH ( below ) . Shown is fold - change compared to shLuc in control media , bars and error bars represent mean and standard deviation of three biological replicates , unpaired t - test ) . Left : Representative immunofluorescence ( IF ) images showing primary cilium formation in RPE cells upon starvation after infection with shLuc ( above ) or shGPR56strong ( below ) . The non - motile primary cilium is visualized using antibodies against γ - tubulin , which stains the basal body , and the ciliary membrane marker ARL13B . DAPI indicates the nucleus . Images on the right show selected cells at 3 x magnification . Brightness was increased in all images to enhance visibility of cilia . Right : Percentage of RPE - 1 cells with primary cilium in shLuc vs . shGPR56strong RPE - 1 cells at different time points after the start of serum starvation . Unpaired t - test , bars and error bars represent mean and stdev of three biological replicate wells . * * * * p < 0 . 0001 , * * * p < 0 . 0005 , * * p < 0 . 005 , * p < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 3  β-catenin protein expression in K562 cells infected with shGPR56strong or shLuc negative control in presence and absence of Wnt3a (representative Western Blot (above) and quantification after normalization to GAPDH (below). Shown is fold-change compared to shLuc in control media, bars and error bars represent mean and standard deviation of three biological replicates, unpaired t-test).  Left: Representative immunofluorescence (IF) images showing primary cilium formation in RPE cells upon starvation after infection with shLuc (above) or shGPR56strong (below). The non-motile primary cilium is visualized using antibodies against γ-tubulin, which stains the basal body, and the ciliary membrane marker ARL13B. DAPI indicates the nucleus. Images on the right show selected cells at 3 x magnification. Brightness was increased in all images to enhance visibility of cilia. Right: Percentage of RPE-1 cells with primary cilium in shLuc vs. shGPR56strong RPE-1 cells at different time points after the start of serum starvation. Unpaired t-test, bars and error bars represent mean and stdev of three biological replicate wells. **** p<0.0001, *** p<0.0005, ** p<0.005, * p<0.05.


Figure 1 ( A ) RT - qPCR analysis of NimB1 , NimB2 , NimB3 , NimB4 and NimB5 transcripts , normalized to RpL32 , from carcass , macrophages , fat body and gut of wild type ( w1118 ) L3 wandering larvae . Data are represented as mean ± SD from three independent experiments . ( B ) Representative confocal imaging of NimB4 - sGFP macrophages of embryo at stage 16 ; lateral ( left ) and ventral ( right ) views ; Tissues were stained with anti - GFP ( green , corresponding to NimB4 - sGFP ) and anti - SIMU antibodies ( red , corresponding to macrophages ) . The arrows show the presence of NimB4 - sGFP inside the embryonic macrophages . Scale bars = 20

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Figure 1(A) RT-qPCR analysis of NimB1, NimB2, NimB3, NimB4 and NimB5 transcripts, normalized to RpL32, from carcass, macrophages, fat body and gut of wild type (w1118) L3 wandering larvae. Data are represented as mean ± SD from three independent experiments.(B) Representative confocal imaging of NimB4-sGFP macrophages of embryo at stage 16; lateral (left) and ventral (right) views; Tissues were stained with anti-GFP (green, corresponding to NimB4-sGFP) and anti-SIMU antibodies (red, corresponding to macrophages). The arrows show the presence of NimB4-sGFP inside the embryonic macrophages. Scale bars = 20 μm


Figure 2 ( A - C ) Representative confocal imaging of CFSE - stained apoptotic bodies ( green ) incubated with secreted NimB4 - RFP ( B , C ) or SPvkg - RFP ( A ) . Apoptotic bodies were pre - incubated in absence ( A , B ) or presence ( C ) of annexin V ( 25 μg / mL ) . Scale bar = 10 μm . ( D ) Quantification of the colocalization of NimB4 - RFP or SPvkg - RFP with the apoptotic bodies in presence or absence of Annexin V , as measured by Pearson ' s correlation coefficient . Values from at least four independent experiments are represented as mean ± SD ( * * P < 0 . 01 by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests . ns : not significant ) .

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Figure 2(A-C) Representative confocal imaging of CFSE-stained apoptotic bodies (green) incubated with secreted NimB4-RFP (B, C) or SPvkg-RFP (A). Apoptotic bodies were pre-incubated in absence (A, B) or presence (C) of annexin V (25 μg/mL). Scale bar = 10 μm. (D) Quantification of the colocalization of NimB4-RFP or SPvkg-RFP with the apoptotic bodies in presence or absence of Annexin V, as measured by Pearson's correlation coefficient. Values from at least four independent experiments are represented as mean ± SD (** P<0.01 by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant).


Figure 3 ( A ) Representative confocal imaging of wild type ( w1118 ) , NimB4 sk2 and apoptosis - null NimB4 sk2 ; Df ( 3L ) H99 embryonic macrophages at stage 16 of development ; ventral and lateral view ; Tissues were co - stained with Dcp - 1 ( red , corresponding to apoptotic corpses ) and anti - SIMU ( green , corresponding to macrophages ) antibodies . The arrows show the presence of apoptotic cells inside embryonic macrophages . Scale bars = 20 μm ( C ) Representative projections from confocal stacks of entire third instar wild type ( w1118 ) and NimB4 sk2 larval brains stained with anti - Draper ( green ) and anti - Dcp1 ( red ) . The arrows show the presence of apoptotic cells in the central area of NimB4 sk2 larval brain . Scale bars = 100

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Figure 3(A) Representative confocal imaging of wild type (w1118), NimB4 sk2 and apoptosis-null NimB4 sk2; Df(3L) H99 embryonic macrophages at stage 16 of development; ventral and lateral view; Tissues were co-stained with Dcp-1 (red, corresponding to apoptotic corpses) and anti-SIMU (green, corresponding to macrophages) antibodies. The arrows show the presence of apoptotic cells inside embryonic macrophages. Scale bars = 20 μm(C) Representative projections from confocal stacks of entire third instar wild type (w1118) and NimB4 sk2 larval brains stained with anti-Draper (green) and anti-Dcp1 (red). The arrows show the presence of apoptotic cells in the central area of NimB4 sk2 larval brain. Scale bars = 100 μm


Figure 4 ( A ) Representative confocal imaging of wild type ( w1118 ) , NimB4 sk2 and draper macrophages incubated with fluorescently - labeled apoptotic cells ( red , CellTraceTM Red ) on ice for 1h and stained with AlexaFluorTM 488 phalloidin ( green ) . Scale bar : 10 μm . ( B ) Quantification of the number of apoptotic cells ( red ) binding to the macrophages ( green ) . Values from three independent experiments are represented as mean ± SD ( * * * P < 0 . 001 by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests . ns : not significant ) . ( D ) Ex vivo phagocytosis assay using Alexa555 fluorescent apoptotic bodies . Wild type , NimB4sk2 , NimB4 genomic rescue ( NimB4sk2 , [ NimB4 ] ) , draper and NimC11 , eater1 macrophages from L3 wandering were incubated with Alexa555 fluorescent apoptotic bodies for 30 , 60 or 120 min at room temperature . Phagocytosis was quantified by flow cytometry . Data are represented as mean ± SD from three independent experiments ( * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests ns : not significant ) . ( E ) Ex vivo phagocytosis assay using AlexFluor488 S . aureus Bioparticles . Wild type , NimB4sk2 and NimC11 , eater1 macrophages from L3 wandering were incubated with bioparticles for 30 , 60 or 120 min at room temperature . Phagocytosis was quantified by flow cytometry . Data are represented as mean ± SD from three independent experiments ( * * * * P < 0 . 0001 by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests . ns : not significant ) .

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Figure 4(A) Representative confocal imaging of wild type (w1118), NimB4 sk2 and draper macrophages incubated with fluorescently-labeled apoptotic cells (red, CellTraceTM Red) on ice for 1h and stained with AlexaFluorTM 488 phalloidin (green). Scale bar: 10 μm. (B) Quantification of the number of apoptotic cells (red) binding to the macrophages (green). Values from three independent experiments are represented as mean ± SD (*** P<0.001 by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant). (D) Ex vivo phagocytosis assay using Alexa555 fluorescent apoptotic bodies. Wild type, NimB4sk2, NimB4 genomic rescue (NimB4sk2, [NimB4]), draper and NimC11, eater1 macrophages from L3 wandering were incubated with Alexa555 fluorescent apoptotic bodies for 30, 60 or 120 min at room temperature. Phagocytosis was quantified by flow cytometry. Data are represented as mean ± SD from three independent experiments (* P<0.05,** P<0.01, **** P<0.0001 by ANOVA test followed by post hoc Dunnett's multiple comparison tests ns: not significant). (E) Ex vivo phagocytosis assay using AlexFluor488 S.aureus Bioparticles. Wild type, NimB4sk2 and NimC11, eater1 macrophages from L3 wandering were incubated with bioparticles for 30, 60 or 120 min at room temperature. Phagocytosis was quantified by flow cytometry. Data are represented as mean ± SD from three independent experiments (**** P<0.0001 by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant).


Figure 5 ( A ) Representative fluorescence microscopy images of wild type ( w1118 ) , NimB4sk2 , draper , eater1 and croquemort △ third instar larvae macrophages stained with LysoTracker Red ( live imaging ) . Overlay of fluorescence and differential interference contrast microscopy ( DIC ) . Scale bar = 10 μm . ( B ) Mean fluorescence intensity after staining with LysoTracker Red ( live confocal imaging ) . Values from at least three independent experiments are represented as mean ± SD ( * * P < 0 . 01 , * * * * P < 0 . 0001 by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests ) . ( C ) Representative transmission electron micrographs of macrophages from wild type ( top , w1118 ) and NimB4sk2 ( bottom ) L3 wandering larvae . Scale bar : 2 μm ( D ) Quantification of the number of vesicles per macrophages in the electron micrographs . Values from at least three independent experiments are represented as mean ± SD ( * * * * P < 0 . 0001 by Mann - Whitney test ) . ( E ) Representative confocal imaging of Rab7EYFP immunostaining in wild type ( w1118 ) and NimB4 sk2 macrophages . Tissues were stained with anti - GFP ( red , Rab7 ) , counterstained with phalloidin ( gray ) and DAPI ( blue ) . The arrows indicate the enlarged vesicles are decorated with Rab7EYFP in the NimB4sk2 macrophages . Scale bar = 10 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 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 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

Figure 5(A) Representative fluorescence microscopy images of wild type (w1118), NimB4sk2, draper, eater1 and croquemort△ third instar larvae macrophages stained with LysoTracker Red (live imaging). Overlay of fluorescence and differential interference contrast microscopy (DIC). Scale bar = 10 μm. (B) Mean fluorescence intensity after staining with LysoTracker Red (live confocal imaging). Values from at least three independent experiments are represented as mean ± SD ( ** P<0.01, **** P<0.0001 by ANOVA test followed by post hoc Dunnett's multiple comparison tests). (C) Representative transmission electron micrographs of macrophages from wild type (top, w1118) and NimB4sk2 (bottom) L3 wandering larvae. Scale bar: 2 μm (D) Quantification of the number of vesicles per macrophages in the electron micrographs. Values from at least three independent experiments are represented as mean ± SD (**** P<0.0001 by Mann-Whitney test). (E) Representative confocal imaging of Rab7EYFP immunostaining in wild type (w1118) and NimB4 sk2 macrophages. Tissues were stained with anti-GFP (red, Rab7), counterstained with phalloidin (gray) and DAPI (blue). The arrows indicate the enlarged vesicles are decorated with Rab7EYFP in the NimB4sk2 macrophages. Scale bar = 10 μm.