Figure 2A . Deconvolved inverted fluorescence micrographs Chm7 - GFP and truncations in the indicated null strains . Scale bar is 5 μm . B . Plot of the percentage of cells with SD of Chm7 - GFP ( and truncations ) NE foci from A . Data are from 3 independent replicates where > 200 cells were counted for each strain . p values from un - paired student ' s T - test . * * is a p value ≤ 0 . 01 ; * * * * , p ≤ 0 . 0001 . C . Deconvolved inverted fluorescence micrographs of Chm7 - GFP in a heh1Δ strain containing either an empty pRS426 plasmid ( - ) or those expressing HEH1 ( pRS426 - HEH1 ) or HEH2 ( pRS426 - HEH2 ) . D . Plots of the percentage of cells from C with Chm7 - GFP NE - foci . Data are from 3 independent replicates where > 200 cells were counted for each strain . Error bars are SD . p values from T - test . * * , p ≤ 0 . 01 ; * * * * , 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 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 2A. Deconvolved inverted fluorescence micrographs Chm7-GFP and truncations in the indicated null strains. Scale bar is 5 μm.B. Plot of the percentage of cells with SD of Chm7-GFP (and truncations) NE foci from A. Data are from 3 independent replicates where >200 cells were counted for each strain. p values from un-paired student's T-test. ** is a p value ≤ 0.01; ****, p ≤ 0.0001.C. Deconvolved inverted fluorescence micrographs of Chm7-GFP in a heh1Δ strain containing either an empty pRS426 plasmid (-) or those expressing HEH1 (pRS426-HEH1) or HEH2 (pRS426-HEH2).D. Plots of the percentage of cells from C with Chm7-GFP NE-foci. Data are from 3 independent replicates where >200 cells were counted for each strain. Error bars are SD. p values from T-test. **, p ≤ 0.01; ****, p ≤ 0.0001.


Figure 3C . GST , GST - Snf7 and GST - snf7OPEN were immobilized on GT - resin and incubated with buffer ( - ) or recombinant heh2 ( 1 - 308 ) - His6 . Bound proteins separated by SDS - PAGE were visualized by coomassie stain and by Western blot with anti - His6 antibody and ECL detection ( WB ; top panel ) . Middle panel shows indicated cropped region of gel where contrast has been increased . Numbers on side of gel show position of molecular weight ( MW ) markers . D . In vitro transcription translation ( IVT ) reactions generating radiolabeled ( 35S ) truncations of Heh2 ( inputs ) were incubated with bead - bound GST , GST - snf7OPEN or GST - Snf7 before washing , elution and detection of bound proteins by autoradiography ( top panel ) or coomassie staining ( bottom ) . E . As in C except with GST - Chm7 constructs . Upper panel shows western blot ( WB ) with anti - His6 antibody . * chm7 - NTD is also expressed with a His6 tag . Bottom panel is coomassie stained . F . As in E except with MBP - Snf7 .

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

Figure 3C. GST, GST-Snf7 and GST-snf7OPEN were immobilized on GT-resin and incubated with buffer(-) or recombinant heh2(1-308)-His6. Bound proteins separated by SDS-PAGE were visualized by coomassie stain and by Western blot with anti-His6 antibody and ECL detection (WB; top panel). Middle panel shows indicated cropped region of gel where contrast has been increased. Numbers on side of gel show position of molecular weight (MW) markers.D. In vitro transcription translation (IVT) reactions generating radiolabeled (35S) truncations of Heh2 (inputs) were incubated with bead-bound GST, GST-snf7OPEN or GST-Snf7 before washing, elution and detection of bound proteins by autoradiography (top panel) or coomassie staining (bottom).E. As in C except with GST-Chm7 constructs. Upper panel shows western blot (WB) with anti-His6 antibody. *chm7-NTD is also expressed with a His6 tag. Bottom panel is coomassie stained.F. As in E except with MBP-Snf7.


Figure 4A . Deconvolved inverted fluorescent micrographs of BiFC signal of Chm7 - VC with either Heh1 - VN or Heh2 - VN . Cell borders are outlined . Scale bar is 5 μm . B . Deconvolved inverted fluorescent micrographs of yeast strains expressing Heh1 - VN and Snf7 - VC with either Chm7 - mCherry or Mps3 - mCherry and merge of both channels . Scale bar is 5 μm . C . Deconvolved inverted fluorescent micrographs of BiFC signal of the indicated VN and VC fusions in the indicated strains . Cell borders outlined . Scale bar is 5 μm . D , E . Plots of the percentage of cells with BiFC signal of the indicated VN VC pairs in the indicated null backgrounds . Data are from three independent replicates where > 225 cells per strain per replicate were quantified . Error bars are SD from the mean of each replicate . p values from un - paired student ' s T - test where ns is p > 0 . 05 . * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 ; * * * * , p ≤ 0 . 0001 . F . Deconvolved inverted fluorescence micrographs of Heh1 - VN Snf7 - VC BiFC signal in a chm7Δ strain with either an empty plasmid ( " - " ; pRS416 ) or one expressing CHM7 ( pRS416 - HA - CHM7 ) . Cell borders outlined . Scale bar is 5 μm . G . Plot of the percentage of cells with BiFC signal from F . Error bars are SD from the mean of each replicate . p values from un - paired student ' s T - test where ns is p > 0 . 05 . * * * , 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 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 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 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 4A. Deconvolved inverted fluorescent micrographs of BiFC signal of Chm7-VC with either Heh1-VN or Heh2-VN. Cell borders are outlined. Scale bar is 5 μm.B. Deconvolved inverted fluorescent micrographs of yeast strains expressing Heh1-VN and Snf7-VC with either Chm7-mCherry or Mps3-mCherry and merge of both channels. Scale bar is 5 μm.C. Deconvolved inverted fluorescent micrographs of BiFC signal of the indicated VN and VC fusions in the indicated strains. Cell borders outlined. Scale bar is 5 μm.D,E. Plots of the percentage of cells with BiFC signal of the indicated VN VC pairs in the indicated null backgrounds. Data are from three independent replicates where > 225 cells per strain per replicate were quantified. Error bars are SD from the mean of each replicate. p values from un-paired student's T-test where ns is p > 0.05. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.F. Deconvolved inverted fluorescence micrographs of Heh1-VN Snf7-VC BiFC signal in a chm7Δ strain with either an empty plasmid ("-"; pRS416) or one expressing CHM7 (pRS416-HA-CHM7). Cell borders outlined. Scale bar is 5 μm.G. Plot of the percentage of cells with BiFC signal from F. Error bars are SD from the mean of each replicate. p values from un-paired student's T-test where ns is p > 0.05. ***, p ≤ 0.001.


Figure 5A . Deconvolved inverted fluorescence micrographs of Chm7 - GFP in the indicated strains . Scale bar is 5 μm . The percentage of cells ± SD with Chm7 - GFP foci is indicated below each panel . B . Deconvolved fluorescence images of SINC - containing nuclei in vps4Δ and vps4Δpom152Δ cells expressing Chm7 - GFP and Nup170 - mCherry ( green , red and merged images are shown ) . Scale bar is 1 μm . C . Plot of the proportion of nup clusters with Chm7 - GFP enrichment in the indicated genetic backgrounds . Error bars are SD of the mean from 3 independent replicates . D . Deconvolved fluorescence micrograph showing the lack of colocalization of Chm7 - GFP and clustered Nup170 - mCherry in a nup133Δ strain . Scale bar is 1 μm . E . Plot of the total fluorescence / integrated density ( in arbitrary units ( A . U . ) ) of Chm7 - GFP within ( and outside of ) the SINC . Error bars represent SD deviation of the mean from > 50 SINC or non - SINC accumulations pooled from 3 independent replicates . p values from un - paired student ' s T - tests with Welch ' s correction . * * * * is p ≤ 0 . 0001 . F . Correlation of the total fluorescence intensity / integrated density of SINC - enriched Chm7 - GFP and Nup170 - mCherry within individual SINCs . Linear regression calculated from 150 SINCs pooled from 3 independent replicates ; r is the linear correlation ( Pearson ' s ) coefficient .

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

Figure 5A. Deconvolved inverted fluorescence micrographs of Chm7-GFP in the indicated strains. Scale bar is 5 μm. The percentage of cells ± SD with Chm7-GFP foci is indicated below each panel.B. Deconvolved fluorescence images of SINC-containing nuclei in vps4Δ and vps4Δpom152Δ cells expressing Chm7-GFP and Nup170-mCherry (green, red and merged images are shown). Scale bar is 1 μm.C. Plot of the proportion of nup clusters with Chm7-GFP enrichment in the indicated genetic backgrounds. Error bars are SD of the mean from 3 independent replicates.D. Deconvolved fluorescence micrograph showing the lack of colocalization of Chm7-GFP and clustered Nup170-mCherry in a nup133Δ strain. Scale bar is 1 μm.E. Plot of the total fluorescence/integrated density (in arbitrary units (A.U.)) of Chm7-GFP within (and outside of) the SINC. Error bars represent SD deviation of the mean from > 50 SINC or non-SINC accumulations pooled from 3 independent replicates. p values from un-paired student's T-tests with Welch's correction. **** is p ≤ 0.0001.F. Correlation of the total fluorescence intensity/integrated density of SINC-enriched Chm7-GFP and Nup170-mCherry within individual SINCs. Linear regression calculated from 150 SINCs pooled from 3 independent replicates; r is the linear correlation (Pearson's) coefficient.


Figure 6A . 10 - fold serial dilutions of the indicated yeast strains were grown on YPD at 23 ° C for 3 days before imaging . C . Deconvolved fluorescence micrographs of Sna3 - mCherry in the indicated genetic backgrounds . Scale bar is 5 μm . D . Deconvolved fluorescence micrographs ( maximum intensity projections of a z - series of images ) of GFP - Nup49 in the indicated yeast strains ( arrows indicate SINCs ) . E . Plot of proportion of cells in the indicated strains with SINCs . Error bars are the SD from the mean from 3 independent replicates quantifying > 300 cells for each strain . p values from un - paired student ' s T - tests where ns is p > 0 . 05 ; * , 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 B-PANEL_START 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

Figure 6A.10-fold serial dilutions of the indicated yeast strains were grown on YPD at 23°C for 3 days before imaging.C. Deconvolved fluorescence micrographs of Sna3-mCherry in the indicated genetic backgrounds. Scale bar is 5 μm.D. Deconvolved fluorescence micrographs (maximum intensity projections of a z-series of images) of GFP-Nup49 in the indicated yeast strains (arrows indicate SINCs).E. Plot of proportion of cells in the indicated strains with SINCs. Error bars are the SD from the mean from 3 independent replicates quantifying >300 cells for each strain. p values from un-paired student's T-tests where ns is p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.


Figure 7A . Deconvolved inverted fluorescent micrographs of the indicated strains expressing Chm7 - GFP ( percentages ± SD of the proportion of cells with Chm7 - GFP foci is at bottom of each panel , see Figure EV5B ) . Scale bar is 5 μm . B . Plot of the percentage of cells with the indicated number of Chm7 - GFP foci . Error bars are SD from the mean from 3 independent replicates of > 50 foci per strain . p values from 2 - way ANOVA where ns represents p > 0 . 05 ; * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ; * * * * , p ≤ 0 . 0001 . C . Deconvolved inverted fluorescencemicrographs of strains expressing Chm7 - GFP after 5 h at 23 ° C ( top ) or 37 ° C ( bottom ) . Scale bar is 5 μm . Percentages ± SD of the proportion of cells with Chm7 - GFP NE foci are shown under each panel ( see Figure EV5F ) . D . Plots of percentage of cells from C with the indicated number of Chm7 - GFP foci at 23 ° C ( left ) and 37 ° C ( right ) . Error bars are SD from the mean from 3 independent replicates of > 50 foci per strain . p values from 2 - way ANOVA where ns is p > 0 . 05 ; * , p ≤ 0 . 05 ; * * * , 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 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

Figure 7A. Deconvolved inverted fluorescent micrographs of the indicated strains expressing Chm7-GFP (percentages ± SD of the proportion of cells with Chm7-GFP foci is at bottom of each panel, see Figure EV5B). Scale bar is 5 μm.B. Plot of the percentage of cells with the indicated number of Chm7-GFP foci. Error bars are SD from the mean from 3 independent replicates of > 50 foci per strain. p values from 2-way ANOVA where ns represents p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ****, p ≤ 0.0001.C. Deconvolved inverted fluorescencemicrographs of strains expressing Chm7-GFP after 5 h at 23°C (top) or 37°C (bottom). Scale bar is 5 μm. Percentages ± SD of the proportion of cells with Chm7-GFP NE foci are shown under each panel (see Figure EV5F).D. Plots of percentage of cells from C with the indicated number of Chm7-GFP foci at 23°C (left) and 37°C (right). Error bars are SD from the mean from 3 independent replicates of > 50 foci per strain. p values from 2-way ANOVA where ns is p > 0.05; *, p ≤ 0.05; ***, p ≤ 0.001.


Figure 8A . Plot of the percentage of nup116Δ cells incubated for 3 h at 37 ° C with nuclear rim - like accumulations of Chm7 - GFP . Error bars are SD from the mean from 3 independent replicates of > 100 cells . p values from unpaired student ' s T - test where * * * is p ≤ 0 . 001 . B . Deconvolved inverted fluorescence micrographs of Chm7 - GFP in nup116Δ cells at either 23 ° C or grown for 3 h at 37 ° C . Percentages ± SD of the proportion of cells with Chm7 - GFP NE foci are shown under each panel ( see Figure EV5F ) . C . Electron micrographs showing herniations of the NE in nup116Δ cells after incubation for 3 h at 37 ° C . N denotes nuclear interior . Scale bars are 100 nm . D . 10 - fold serial dilutions of indicated strains grown in medium permissive ( - URA ) or restrictive ( 5 - FOA ) of the retention of a URA3 / NUP116 plasmid . Images acquired after incubation for 3 days ( left ) or 6 days ( right ) at 23 ° C .

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 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 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

Figure 8A. Plot of the percentage of nup116Δ cells incubated for 3 h at 37°C with nuclear rim-like accumulations of Chm7-GFP. Error bars are SD from the mean from 3 independent replicates of >100 cells. p values from unpaired student's T-test where *** is p ≤ 0.001.B. Deconvolved inverted fluorescence micrographs of Chm7-GFP in nup116Δ cells at either 23°C or grown for 3 h at 37°C. Percentages ± SD of the proportion of cells with Chm7-GFP NE foci are shown under each panel (see Figure EV5F).C. Electron micrographs showing herniations of the NE in nup116Δ cells after incubation for 3 h at 37°C. N denotes nuclear interior. Scale bars are 100 nm.D. 10-fold serial dilutions of indicated strains grown in medium permissive (-URA) or restrictive (5-FOA) of the retention of a URA3/NUP116 plasmid. Images acquired after incubation for 3 days (left) or 6 days (right) at 23°C.


Figure 9A . Deconvolved fluorescence micrographs of NLS - GFP in the indicated yeast strains at either 30 ° C or after 2 h at 37 ° C . Scale bar is 5 μm . B . Plot of the mean nuclear to cytosolic fluorescence intensity ratio of NLS - GFP from cells represented in A . Error bars are SD from the mean from 3 independent replicates of 150 cells from each strain . p values from un - paired student ' s T - test where ns is p > 0 . 05 ; * * , p ≤ 0 . 01 ; * * * * , p ≤ 0 . 0001 . Only chm7Δ and chm7Δapq12Δ cells have N : C ratios below 1 . 2 ( dotted red line ) . C . Re - plotting of data from B showing the percentage of cells with a N : C NLS - GFP fluorescence ratio < 1 . 2 . Error bars are the SD from the mean from 3 independent replicates of 150 cells / strain . p values from student ' s T - test where * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ; * * * * , 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 O O O O O O O O O O O O O O O O O O

Figure 9A. Deconvolved fluorescence micrographs of NLS-GFP in the indicated yeast strains at either 30°C or after 2 h at 37°C. Scale bar is 5 μm.B. Plot of the mean nuclear to cytosolic fluorescence intensity ratio of NLS-GFP from cells represented in A. Error bars are SD from the mean from 3 independent replicates of 150 cells from each strain. p values from un-paired student's T-test where ns is p > 0.05; **, p ≤ 0.01; ****, p ≤ 0.0001. Only chm7Δ and chm7Δapq12Δ cells have N:C ratios below 1.2 (dotted red line).C. Re-plotting of data from B showing the percentage of cells with a N:C NLS-GFP fluorescence ratio <1.2. Error bars are the SD from the mean from 3 independent replicates of 150 cells/strain. p values from student's T-test where *, p ≤ 0.05; **, p ≤ 0.01; ****, p ≤ 0.0001.


Figure 1 ( B ) High magnification view of boxed regions in ( A ) pseudo - colored to show actin depletion in anaphase . Scale bar - 1 µm . ( F ) Stills from time - lapse sequence of representative Hela cells expressing mNeon - Green Aurora B and Cell Mask to label cell membrane , exiting mitosis , showing the dynamic re - localization of Aurora B from DNA to the overlapping microtubules and cleavage furrow with control siRNA treatment ( arrows top ) . With knock - down of RACGAP1 , Aurora B still re - localises from the DNA to the microtubules and furrow in the midzone in early anaphase ( arrows bottom ) , but the microtubule localization is lost at later stages ( asterisk bottom ) . 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 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 1(B) High magnification view of boxed regions in (A) pseudo-colored to show actin depletion in anaphase. Scale bar -1 µm.(F) Stills from time-lapse sequence of representative Hela cells expressing mNeon-Green Aurora B and Cell Mask to label cell membrane, exiting mitosis, showing the dynamic re-localization of Aurora B from DNA to the overlapping microtubules and cleavage furrow with control siRNA treatment (arrows top). With knock-down of RACGAP1, Aurora B still re-localises from the DNA to the microtubules and furrow in the midzone in early anaphase (arrows bottom), but the microtubule localization is lost at later stages (asterisk bottom). Scale bar - 10 µm.


Figure 2 ( E ) Stills of representative Hela cells expressing Lifeact - GFP and H2B - mCherry at 10 mins following forced mitotic exit with siControl , siRACGAP1 , DMSO and 2µM ZM447439 treatment . Control siRNA , RACGAP1 siRNA and DMSO treated cells show clearance of actin from the cortex close to the DNA , while ZM447439 treated cells fail to clear actin , quantified in ( F ) . Note , failure in actin accumulation in RACGAP1 depleted cell , asterisk . Scale bar - 10 µm ( F ) Quantification of actin intensity across the perimeter of the cells based on distance from DNA , with 5 and 6 being closest to DNA and 1 and 10 furthest away , as in cartoon ( B ) . Graph shows decrease in actin intensity closer to the DNA in siControl ( n = 26 ) , siRACGAP1 ( n = 37 ) and Control DMSO treated cells ( n = 38 cells ) , seen by levels lower than 1 ( dotted red line ) , while this decrease is absent in ZM447439 treated cells ( n = 42 cells ) . Data presented as mean ± SD .

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

Figure 2(E) Stills of representative Hela cells expressing Lifeact-GFP and H2B-mCherry at 10 mins following forced mitotic exit with siControl, siRACGAP1, DMSO and 2µM ZM447439 treatment. Control siRNA, RACGAP1 siRNA and DMSO treated cells show clearance of actin from the cortex close to the DNA, while ZM447439 treated cells fail to clear actin, quantified in (F). Note, failure in actin accumulation in RACGAP1 depleted cell, asterisk. Scale bar - 10 µm(F) Quantification of actin intensity across the perimeter of the cells based on distance from DNA, with 5 and 6 being closest to DNA and 1 and 10 furthest away, as in cartoon (B). Graph shows decrease in actin intensity closer to the DNA in siControl (n=26), siRACGAP1 (n=37) and Control DMSO treated cells (n=38 cells), seen by levels lower than 1 (dotted red line), while this decrease is absent in ZM447439 treated cells (n=42 cells). Data presented as mean ± SD.


Figure 3 ( B ) Stills from time - lapse sequence of the basal most region of Hela cells expressing Lifeact - GFP and H2B - mCherry forced to undergo flat monopolar mitotic exit . While actin is cleared underneath the DNA in control DMSO treated cells , it fails to be cleared around the DNA when cells are treated with Aurora B inhibitor ZM447439 during mitotic exit . Dotted mask shows position of DNA in actin channel . Scale bar - 10µm . ( C ) Quantification of clearance of actin underneath the DNA for still shown in ( B ) . While DNA under control cell starts clearing actin within 10 mins , Aurora B inhibited cells fail to do so .

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Figure 3(B) Stills from time-lapse sequence of the basal most region of Hela cells expressing Lifeact-GFP and H2B-mCherry forced to undergo flat monopolar mitotic exit. While actin is cleared underneath the DNA in control DMSO treated cells, it fails to be cleared around the DNA when cells are treated with Aurora B inhibitor ZM447439 during mitotic exit. Dotted mask shows position of DNA in actin channel. Scale bar -10µm.(C) Quantification of clearance of actin underneath the DNA for still shown in (B). While DNA under control cell starts clearing actin within 10 mins, Aurora B inhibited cells fail to do so.


Figure 4 ( D ) Still images of representative Hela cells expressing Lifeact - GFP and H2B - mCherry captured at early anaphase under different conditions i . e . Control siRNA , knock - down of centralspindlin protein RACGAP1 , in the presence and absence of Aurora B kinase inhibitor ( 2µM ZM ) . Scale bar - 10 µm ( E ) Quantification of aspect ratio of cells treated with different conditions as in ( D ) at 6 mins after anaphase onset , before furrow formation . Aurora B inhibition and siRACGAP1 individually have a moderate effect on aspect ratio , while together have a stronger impact during mitotic exit . In combined treatment , mitotic cells hardly undergo any elongation and remain spherical before re - spreading . Ordinary one - way ANOVA with multiple comparison . Each treatment is compared with siControl , * * * * p < 0 . 0001 for all comparisons . Data represented as box - whisker plot , with box showing 25th - 75th percentile values , whiskers - min to max value and line representing median . Mean ± SD for different treatments - siControl ( n = 38 ) - 1 . 455 ± 0 . 19 , ZM ( n = 32 ) - 1 . 219 ± 0 . 16 , siRACGAP1 ( n = 25 ) - 1 . 144 ± 0 . 078 and siRACGAP1 + ZM ( n = 16 ) - 1 . 013 ± 0 . 03 . Cells for different treatment pooled from n > 3 independent experiments .

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Figure 4(D) Still images of representative Hela cells expressing Lifeact-GFP and H2B-mCherry captured at early anaphase under different conditions i.e. Control siRNA, knock-down of centralspindlin protein RACGAP1, in the presence and absence of Aurora B kinase inhibitor (2µM ZM). Scale bar - 10 µm (E) Quantification of aspect ratio of cells treated with different conditions as in (D) at 6 mins after anaphase onset, before furrow formation. Aurora B inhibition and siRACGAP1 individually have a moderate effect on aspect ratio, while together have a stronger impact during mitotic exit. In combined treatment, mitotic cells hardly undergo any elongation and remain spherical before re-spreading. Ordinary one-way ANOVA with multiple comparison. Each treatment is compared with siControl, ****p<0.0001 for all comparisons. Data represented as box-whisker plot, with box showing 25th-75th percentile values, whiskers-min to max value and line representing median. Mean±SD for different treatments- siControl (n=38) - 1.455±0.19, ZM (n=32) - 1.219±0.16, siRACGAP1 (n=25) -1.144±0.078 and siRACGAP1+ZM (n=16) - 1.013±0.03. Cells for different treatment pooled from n>3 independent experiments.


Figure 5 ( C ) Quantification of actin intensity at the poles of cells at 6 mins post anaphase onset , before furrow formation under different conditions as in ( A ) . Actin fails to be cleared from the poles upon Aurora B kinase inhibition during mitotic exit and is stronger when both centralspindlin and Aurora B kinase activity are affected , while it is cleared upon RACGAP1 depletion . Data represented as mean ± SD for treatments - siControl - 0 . 98 ± 0 . 05 , siRACGAP1 - 0 . 99 ± 0 . 04 , ZM - 1 ± 0 . 039 , siRACGAP1 + ZM - 1 ± 0 . 037 . Ordinary one - way ANOVA with multiple comparison - siControl vs siRACGAP1 - n . s . , p = 0 . 896 , siControl vs ZM - * * p = 0 . 0018 , siControl vs siRACGAP1 + ZM - * * * * p < 0 . 0001 and ZM vs siRACGAP1 + ZM - p = 0 . 1528 . ( G ) Quantification of shortest distance between centroid of DNA and cortical actomyosin network . While chromatin is able to reach close to the cortex in both siControl and siRACGAP1 treated cells , the distance of chromatin from cortex increases upon ZM treatment and is further enhanced in the double treatment condition . Data represented as mean ± SD . siControl - 4 . 7 ± 0 . 87 , siRACGAP1 - 4 . 4 ± 0 . 64 , ZM - 5 . 47 ± 0 . 87 , siRACGAP1 + ZM - 6 . 35 ± 1 . 14 microns . Ordinary one - way ANOVA with multiple comparison , siControl vs ZM , * * * p = 0 . 002 , siControl vs siRACGAP1 , p = 0 . 2823 , siControl vs siRACGAP1 + ZM , * * * * p < 0 . 0001 , siRACGAP1 vs siRACGAP1 + ZM , * * * * p < 0 . 0001 , ZM vs siRACGAP1 , * * * * p < 0 . 0001 , ZM vs siRACGAP1 + ZM , * * * p = 0 . 0004 .

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 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

Figure 5(C) Quantification of actin intensity at the poles of cells at 6 mins post anaphase onset, before furrow formation under different conditions as in (A). Actin fails to be cleared from the poles upon Aurora B kinase inhibition during mitotic exit and is stronger when both centralspindlin and Aurora B kinase activity are affected, while it is cleared upon RACGAP1 depletion. Data represented as mean±SD for treatments - siControl- 0.98±0.05, siRACGAP1 - 0.99±0.04, ZM - 1±0.039, siRACGAP1+ZM - 1±0.037. Ordinary one-way ANOVA with multiple comparison- siControl vs siRACGAP1 - n.s., p=0.896, siControl vs ZM - **p=0.0018, siControl vs siRACGAP1+ZM - ****p<0.0001 and ZM vs siRACGAP1+ZM - p=0.1528.(G) Quantification of shortest distance between centroid of DNA and cortical actomyosin network. While chromatin is able to reach close to the cortex in both siControl and siRACGAP1 treated cells, the distance of chromatin from cortex increases upon ZM treatment and is further enhanced in the double treatment condition. Data represented as mean±SD. siControl - 4.7±0.87, siRACGAP1 - 4.4±0.64, ZM - 5.47±0.87, siRACGAP1+ZM - 6.35±1.14 microns. Ordinary one-way ANOVA with multiple comparison, siControl vs ZM, ***p=0.002, siControl vs siRACGAP1, p=0.2823, siControl vs siRACGAP1+ZM, ****p<0.0001, siRACGAP1 vs siRACGAP1+ZM, ****p <0.0001, ZM vs siRACGAP1, ****p<0.0001, ZM vs siRACGAP1+ZM, ***p=0.0004 .


Figure 1Differences in clinical variables including ( B ) liver fat , plasma levels of ( C ) ALT , ( D ) AST , ( E ) uric acid and ( F ) creatinine are presented in the CMA and placebo groups on Days 0 , 14 and 70 after weight loss adjustment . Adj . P indicates p value after weight loss adjustment . Statistical significance is defined based on paired Student ' s t test . p < 0 . 05 . The boxes show the distribution of the clinical parameters in different groups . The bottom and top of the boxes represent the 25th and 75th percentiles . The central band represents the median value . The whiskers represent the minimum and maximum values that are not outliers and dots represent outlier values . The sample sizes on Days 0 , 14 or 70 were marked in each boxplot .

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Figure 1Differences in clinical variables including (B) liver fat, plasma levels of (C) ALT, (D) AST, (E) uric acid and (F) creatinine are presented in the CMA and placebo groups on Days 0, 14 and 70 after weight loss adjustment. Adj.P indicates p value after weight loss adjustment. Statistical significance is defined based on paired Student's t test. p< 0.05. The boxes show the distribution of the clinical parameters in different groups. The bottom and top of the boxes represent the 25th and 75th percentiles. The central band represents the median value. The whiskers represent the minimum and maximum values that are not outliers and dots represent outlier values. The sample sizes on Days 0, 14 or 70 were marked in each boxplot.


Figure 2Differences in clinical variables including ( B ) liver fat , plasma levels of ( C ) ALT , ( D ) AST , ( E ) uric acid and ( F ) creatinine are presented in the CMA and placebo groups on Days 0 , 14 and 70 after weight loss adjustment . Adj . P indicates p value after weight loss adjustment . Statistical significance is defined based on paired Student ' s t test . p < 0 . 05 . The boxes show the distribution of the clinical parameters in different groups . The bottom and top of the boxes represent the 25th and 75th percentiles . The central band represents the median value . The whiskers represent the minimum and maximum values that are not outliers and dots represent outlier values . The sample sizes on Days 0 , 14 or 70 were marked in each boxplot .

Figure 2Differences in clinical variables including (B) liver fat, plasma levels of (C) ALT, (D) AST, (E) uric acid and (F) creatinine are presented in the CMA and placebo groups on Days 0, 14 and 70 after weight loss adjustment. Adj.P indicates p value after weight loss adjustment. Statistical significance is defined based on paired Student's t test. p< 0.05. The boxes show the distribution of the clinical parameters in different groups. The bottom and top of the boxes represent the 25th and 75th percentiles. The central band represents the median value. The whiskers represent the minimum and maximum values that are not outliers and dots represent outlier values. The sample sizes on Days 0, 14 or 70 were marked in each boxplot.


Figure 3Association between the plasma level of significantly different metabolites on Day 70 vs Day 0 B ) in both ( n = 4 ) ;

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Figure 3Association between the plasma level of significantly different metabolites on Day 70 vs Day 0 B) in both (n=4);


Figure 4A ) Heatmap shows log2FC based alterations between the significantly different inflammation related proteins on Day 70 vs Day 0 in the CMA and placebo groups . Asterisks indicate statistical significance based on paired Student ' s t test . p < 0 . 05 ; B ) Heatmap shows the correlation between the plasma levels of all inflammation related proteins and plasma levels of the individual metabolic activators including serine , cysteine , carnitine and nicotinamide . Asterisks indicate statistical significance based on Spearman correlation analysis . p < 0 . 05 ; Heatmap shows the associations between the significantly different inflammation related proteins ( CD8A , CSF - 1 , CCL23 , FGF - 21 and OSM ) Asterisks indicate statistical significance based on Spearman correlation analysis . p < 0 . 05 ; Heatmap shows the associations between the significantly different inflammation related proteins ( CD8A , CSF - 1 , CCL23 , FGF - 21 and OSM ) C ) with the 10 most significantly correlated plasma metabolites ,

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Figure 4A) Heatmap shows log2FC based alterations between the significantly different inflammation related proteins on Day 70 vs Day 0 in the CMA and placebo groups. Asterisks indicate statistical significance based on paired Student's t test. p < 0.05;B) Heatmap shows the correlation between the plasma levels of all inflammation related proteins and plasma levels of the individual metabolic activators including serine, cysteine, carnitine and nicotinamide. Asterisks indicate statistical significance based on Spearman correlation analysis. p < 0.05; Heatmap shows the associations between the significantly different inflammation related proteins (CD8A, CSF-1, CCL23, FGF-21 and OSM)Asterisks indicate statistical significance based on Spearman correlation analysis. p < 0.05; Heatmap shows the associations between the significantly different inflammation related proteins (CD8A, CSF-1, CCL23, FGF-21 and OSM) C) with the 10 most significantly correlated plasma metabolites,


Figure 5Scatter plot with linear regression line and heatmap show log2FC based alterations of the significantly different species in the A ) gut microbiome between CMA and placebo groups on Day 70 vs Day 0 ; B ) gut microbiome between Day 70 vs Day 0 in the CMA and placebo groups ; C ) oral microbiome between CMA and placebo groups on Day 70 vs Day 0 ; D ) oral microbiome between Day 70 vs Day 0 in the CMA and placebo groups . Each dot represents a species and it has been colored according to its corresponding phylum . Asterisks indicate statistical significance based on paired Wilcoxon signed - rank test . p < 0 . 05 . Log2FC : log2 ( fold change ) .

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Figure 5Scatter plot with linear regression line and heatmap show log2FC based alterations of the significantly different species in the A) gut microbiome between CMA and placebo groups on Day 70 vs Day 0; B) gut microbiome between Day 70 vs Day 0 in the CMA and placebo groups; C) oral microbiome between CMA and placebo groups on Day 70 vs Day 0; D) oral microbiome between Day 70 vs Day 0 in the CMA and placebo groups. Each dot represents a species and it has been colored according to its corresponding phylum. Asterisks indicate statistical significance based on paired Wilcoxon signed-rank test. p< 0.05. Log2FC: log2(fold change).


Figure 6Heatmap shows the association between the plasma level of clinical variables including liver fat , ALT , AST , uric acid , and creatinine with , B ) plasma level of 10 most significant inflammation related proteinsHeatmap shows the association between the plasma level of clinical variables including liver fat , ALT , AST , uric acid , and creatinine with , C ) the abundance of the species in gut microbiomeHeatmap shows the association between the plasma level of clinical variables including liver fat , ALT , AST , uric acid , and creatinine with D ) the abundance of the species in oral microbiome . AST , Aspartate aminotransferase ; ALT , Alanine transaminase . Asterisks indicate statistical significance based on Spearman correlation analysis . p < 0 . 05 ; Cor . Coeff : Correlation coefficient .

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Figure 6Heatmap shows the association between the plasma level of clinical variables including liver fat, ALT, AST, uric acid, and creatinine with , B) plasma level of 10 most significant inflammation related proteinsHeatmap shows the association between the plasma level of clinical variables including liver fat, ALT, AST, uric acid, and creatinine with , C) the abundance of the species in gut microbiomeHeatmap shows the association between the plasma level of clinical variables including liver fat, ALT, AST, uric acid, and creatinine with D) the abundance of the species in oral microbiome. AST, Aspartate aminotransferase; ALT, Alanine transaminase. Asterisks indicate statistical significance based on Spearman correlation analysis. p < 0.05; Cor.Coeff: Correlation coefficient.


Figure 1A , B ( A ) Confocal imaging of FOS DNA FISH with concurrent SPT5 and Pol II - S5P IF showing that SPT5 and Pol II - S5P occupied the FOS loci after serum starvation . Zoomed - in views of the merged regions are indicated by the white arrow . ( B ) Same as Fig 1A . but for FOS DNA FISH with concurrent SPT5 and NELFE IF . E Confocal images showing that SPT5 , NELFA , Pol II - S5P and AFF4 formed nuclear puncta in HCT 116 cells before and after treated with 1 % 1 , 6 - hexanediol ( abbreviation as 1 , 6 - hex ) for 30 minutes . F Violin plot showing the number of cells containing nuclear puncta in Fig 1E . Results are representative of three biological replicates , each n > 20 . Two - tailed , unpaired Student ' s t test was performed . For SPT5 , p = 0 . 1301 . For NELFA , p = 0 . 2756 . For Pol II - S5P , p = 0 . 8179 . For AFF4 , * * * p < 0 . 0001 . GG - I ChIP - qPCR showing that the occupancies of Pol II ( G ) , SPT5 ( H ) , and NELFE ( I ) at the promoters and gene bodies of HSP70 , GAPDH , and MYC in HeLa cells . The HEMO gene serves as a negative control for ChIP - qPCR . Error bars represent standard error of means . Results are plotted with four technical repeats from two biological replicates . Two - tailed , unpaired Student ' s t test was performed . For Pol II ChIP , HSP70 _ promoter , * * p = 0 . 0014 ; GAPDH _ promoter , p = 0 . 8385 ; GAPDH _ gene body , * * * p < 0 . 0001 ; MYC _ promoter , p = 0 . 1765 ; MYC _ gene body , * * * p = 0 . 0002 . For SPT5 ChIP , HSP70 _ promoter , * * p = 0 . 0033 ; GAPDH _ promoter , p = 0 . 9603 ; GAPDH _ gene body , * * p = 0 . 0012 ; MYC _ promoter , p = 0 . 9898 ; MYC _ gene body , * * * p < 0 . 0001 . For NELFE ChIP , HSP70 _ promoter , p = 0 . 3913 ; GAPDH _ promoter , p = 0 . 9347 ; MYC _ promoter , p = 0 . 8558 .

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 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 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

Figure 1A, B (A) Confocal imaging of FOS DNA FISH with concurrent SPT5 and Pol II-S5P IF showing that SPT5 and Pol II-S5P occupied the FOS loci after serum starvation. Zoomed-in views of the merged regions are indicated by the white arrow. (B) Same as Fig 1A. but for FOS DNA FISH with concurrent SPT5 and NELFE IF.E Confocal images showing that SPT5, NELFA, Pol II-S5P and AFF4 formed nuclear puncta in HCT 116 cells before and after treated with 1% 1,6-hexanediol (abbreviation as 1,6-hex) for 30 minutes. F Violin plot showing the number of cells containing nuclear puncta in Fig 1E. Results are representative of three biological replicates, each n > 20. Two-tailed, unpaired Student's t test was performed. For SPT5, p = 0.1301. For NELFA, p = 0.2756. For Pol II-S5P, p = 0.8179. For AFF4, ***p < 0.0001. GG-I ChIP-qPCR showing that the occupancies of Pol II (G), SPT5 (H), and NELFE (I) at the promoters and gene bodies of HSP70, GAPDH, and MYC in HeLa cells. The HEMO gene serves as a negative control for ChIP-qPCR. Error bars represent standard error of means. Results are plotted with four technical repeats from two biological replicates. Two-tailed, unpaired Student's t test was performed. For Pol II ChIP, HSP70_promoter, **p = 0.0014; GAPDH_promoter, p = 0.8385; GAPDH_gene body, ***p < 0.0001; MYC_promoter, p = 0.1765; MYC_gene body, ***p = 0.0002. For SPT5 ChIP, HSP70_promoter, **p = 0.0033; GAPDH_promoter, p = 0.9603; GAPDH_gene body, **p = 0.0012; MYC_promoter, p = 0.9898; MYC_gene body, ***p < 0.0001. For NELFE ChIP, HSP70_promoter, p = 0.3913; GAPDH_promoter, p = 0.9347; MYC_promoter, p = 0.8558.


Figure 2C Normalized FRAP curves for eGFP - SPT5 . The bleaching events occurred at 0 second . Confocal images were embedded within the plot showing FRAP of the in vitro eGFP - SPT5 clusters . Results shown are from three biological replicates . The arrowhead represents the bleached region . E Live cell imaging of HEK - 293T cells expressing eGFP - SPT5 together with mCherry - SPT5 , mCherry - SPT5ΔNTR , or mCherry - SPT5ΔCTR close to endogenous level . F Violin plot showing the clusters area ( top ) and circularity ( bottom ) of SPT5 with SPT5 , SPT5ΔNTR , or SPT5ΔCTR in Fig 2E . Each n > 20 . Results are representative of three biological replicates , each n > 20 . Two - tailed , unpaired Student ' s t test was performed . * * * p < 0 . 001 . GH Confocal images showing FRAP analysis of the NELFA - IDR - eGFP and SPT5 - CTR - mCherry clusters in vitro . The arrowhead represents the bleached and recovered cluster . I Normalized FRAP curves for NELFA - IDR - eGFP and SPT5 - CTR - mCherry clusters . The bleaching events occurred at 0 second . Results shown are from two biological replicates .

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Figure 2C Normalized FRAP curves for eGFP-SPT5. The bleaching events occurred at 0 second. Confocal images were embedded within the plot showing FRAP of the in vitro eGFP-SPT5 clusters. Results shown are from three biological replicates. The arrowhead represents the bleached region.E Live cell imaging of HEK-293T cells expressing eGFP-SPT5 together with mCherry-SPT5, mCherry-SPT5ΔNTR, or mCherry-SPT5ΔCTR close to endogenous level. F Violin plot showing the clusters area (top) and circularity (bottom) of SPT5 with SPT5, SPT5ΔNTR, or SPT5ΔCTR in Fig 2E. Each n > 20. Results are representative of three biological replicates, each n > 20. Two-tailed, unpaired Student's t test was performed. ***p < 0.001. GH Confocal images showing FRAP analysis of the NELFA-IDR-eGFP and SPT5-CTR-mCherry clusters in vitro. The arrowhead represents the bleached and recovered cluster. I Normalized FRAP curves for NELFA-IDR-eGFP and SPT5-CTR-mCherry clusters. The bleaching events occurred at 0 second. Results shown are from two biological replicates.


Figure 3A Western blot analysis of re - expression of mCherry - SPT5 , mCherry - SPT5ΔNTR or mCherry - SPT5ΔCTR after SPT5 depletion . α - Tubulin was used as a loading control . Results are representative of two biological replicates . B Confocal imaging of nascent RNA labeled by EU in HEK - 293T cells expressing SPT5 , SPT5ΔNTR or SPT5ΔCTR after SPT5 depletion . G , H ChIP - qPCR showing the occupancies of Pol II at the promoters and gene bodies of GAPDH ( G ) , and MYC ( H ) in HEK - 293T cells expressing SPT5 , SPT5ΔNTR or SPT5ΔCTR after SPT5 depletion . The HEMO gene serves as a negative control for ChIP - qPCR . Error bars represent standard error of means . Results are plotted with four technical repeats from two biological replicates . Two - tailed , unpaired Student ' s t test was performed . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . = not significant . For GAPDH _ promoter , p value ( from left to right , < 0 . 0001 , < 0 . 0001 , < 0 . 0001 , 0 . 0044 , 0 . 8873 , 0 . 9310 , 0 . 6855 , 0 . 9615 ) ; GAPDH _ gene body , p value ( from left to right , 0 . 0370 , 0 . 0214 , 0 . 0574 , 0 . 0256 , 0 . 9536 , 0 . 8038 , 0 . 7623 , 0 . 8773 ) ; For MYC _ promoter , p value ( from left to right , 0 . 0002 , 0 . 0014 , 0 . 0004 , 0 . 0048 , 0 . 0670 , 0 . 7501 , 0 . 3482 , 0 . 3284 ) ; MYC _ gene body , p value ( from left to right , 0 . 0004 , 0 . 0013 , 0 . 0081 , 0 . 0299 , 0 . 1332 , 0 . 5839 , 0 . 4404 , 0 . 6170 ) .

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 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 O 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 Western blot analysis of re-expression of mCherry-SPT5, mCherry-SPT5ΔNTR or mCherry-SPT5ΔCTR after SPT5 depletion. α-Tubulin was used as a loading control. Results are representative of two biological replicates.B Confocal imaging of nascent RNA labeled by EU in HEK-293T cells expressing SPT5, SPT5ΔNTR or SPT5ΔCTR after SPT5 depletion.G, H ChIP-qPCR showing the occupancies of Pol II at the promoters and gene bodies of GAPDH (G), and MYC (H) in HEK-293T cells expressing SPT5, SPT5ΔNTR or SPT5ΔCTR after SPT5 depletion. The HEMO gene serves as a negative control for ChIP-qPCR. Error bars represent standard error of means. Results are plotted with four technical repeats from two biological replicates. Two-tailed, unpaired Student's t test was performed. *p < 0.05, **p < 0.01, ***p < 0.001, n.s.=not significant. For GAPDH_promoter, p value (from left to right, < 0.0001, < 0.0001, < 0.0001, 0.0044, 0.8873, 0.9310, 0.6855, 0.9615); GAPDH_gene body, p value (from left to right, 0.0370, 0.0214, 0.0574, 0.0256, 0.9536, 0.8038, 0.7623, 0.8773); For MYC_promoter, p value (from left to right, 0.0002, 0.0014, 0.0004, 0.0048, 0.0670, 0.7501, 0.3482, 0.3284); MYC_gene body, p value (from left to right, 0.0004, 0.0013, 0.0081, 0.0299, 0.1332, 0.5839, 0.4404, 0.6170).


Figure 4B Fluorescence microscopy images showing the phase - separated AFF4 - IDR - eGFP and SPT5 - CTR - mCherry droplets formed in 50 mM NaCl containing buffer in the presence of 10 % PEG - 8000 . The concentration ratios are indicated as CAFF4 - IDR / CSPT5 - CTR . C Dot plot showing the droplet area in Fig 4B . Fields per condition n = 5 . DD Live cell confocal images showing FRAP of the heterotypic eGFP - AFF4 + ENL and mCherry - SPT5 droplets . The arrowhead represents the bleached and recovered droplet . E Normalized FRAP curves for eGFP - AFF4 + ENL ( green ) and mCherry - SPT5 ( purple ) in the AFF4 / ENL / SPT5 heterotypic droplets . The bleaching events occurred at 0 second . Results shown are from three biological replicates .

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Figure 4B Fluorescence microscopy images showing the phase-separated AFF4-IDR-eGFP and SPT5-CTR-mCherry droplets formed in 50 mM NaCl containing buffer in the presence of 10% PEG-8000. The concentration ratios are indicated as CAFF4-IDR/CSPT5-CTR. C Dot plot showing the droplet area in Fig 4B. Fields per condition n = 5. DD Live cell confocal images showing FRAP of the heterotypic eGFP-AFF4 + ENL and mCherry-SPT5 droplets. The arrowhead represents the bleached and recovered droplet. E Normalized FRAP curves for eGFP-AFF4 + ENL (green) and mCherry-SPT5 (purple) in the AFF4/ENL/SPT5 heterotypic droplets. The bleaching events occurred at 0 second. Results shown are from three biological replicates.


Figure 5C Confocal imaging of FOS RNA FISH with concurrent SPT5 IF in wild type ( WT ) and ENL KO HCT116 cells after serum treatment . Zoomed - in views of the merged regions are indicated by the white arrow . H Confocal images showing SPT5 in nuclear puncta in WT or ENL KO cells treatment with KL - 1 . I Violin plot showing the number of cells containing nuclear puncta in Fig 5H . Results are representative of three biological replicates , each n > 20 . Two - tailed , unpaired Student ' s t test was performed . * * * p < 0 . 001 . J - M ( J ) Confocal images showing co - localization of Pol II - S5P with SPT5 in nuclear puncta in KL - 1 treated HCT 116 cells . Results shown are representative images from three independent experiments . ( K ) Boxplots showing the mean values of the Pearson correlation coefficient of co - localization ratios of SPT5 and Pol II - S5P in Fig 5J . Central band is median ; boxes represent 1st and 3rd quartile ( 25th and 75th percentile , respectively ) and whiskers 1 . 5 × interquartile range . Results are representative of three biological replicates , each n > 20 . Two - tailed , unpaired Student ' s t test was performed . KL - 1 vs . DMSO , * * p = 0 . 0050 . ( L ) Same as Fig 5J . but for SPT5 and NELFA . ( M ) Same as Fig 5K . but for SPT5 and NELFA . Two - tailed , unpaired Student ' s t test was performed . KL - 1 vs . DMSO , * p = 0 . 0100 .

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

Figure 5C Confocal imaging of FOS RNA FISH with concurrent SPT5 IF in wild type (WT) and ENL KO HCT116 cells after serum treatment. Zoomed-in views of the merged regions are indicated by the white arrow.H Confocal images showing SPT5 in nuclear puncta in WT or ENL KO cells treatment with KL-1. I Violin plot showing the number of cells containing nuclear puncta in Fig 5H. Results are representative of three biological replicates, each n > 20. Two-tailed, unpaired Student's t test was performed. ***p < 0.001.J-M (J) Confocal images showing co-localization of Pol II-S5P with SPT5 in nuclear puncta in KL-1 treated HCT 116 cells. Results shown are representative images from three independent experiments. (K) Boxplots showing the mean values of the Pearson correlation coefficient of co-localization ratios of SPT5 and Pol II-S5P in Fig 5J. Central band is median; boxes represent 1st and 3rd quartile (25th and 75th percentile, respectively) and whiskers 1.5× interquartile range. Results are representative of three biological replicates, each n > 20. Two-tailed, unpaired Student's t test was performed. KL-1 vs. DMSO, **p = 0.0050. (L) Same as Fig 5J. but for SPT5 and NELFA. (M) Same as Fig 5K. but for SPT5 and NELFA. Two-tailed, unpaired Student's t test was performed. KL-1 vs. DMSO, *p = 0.0100.


Figure 6B Fluorescence microscopy images showing that 2 μM SPT5 - CTR - eGFP formed phase - separated droplets in the NaCl containing buffer . C Droplet number ( top ) and droplet area ( bottom ) analyses are shown . Central band is median ; boxes represent 1st and 3rd quartile ( 25th and 75th percentile , respectively ) and whiskers 1 . 5 × interquartile range . Results are representative of two biological replicates , each n > 6 . Two - tailed , unpaired Student ' s t test was performed . For droplet number , SPT5 - CTR + P - TEFb ( - ATP ) vs . SPT5 - CTR , p = * * 0 . 0015 . SPT5 - CTR + P - TEFb ( + vs . - ATP ) , * * p = 0 . 0032 . For droplet area , SPT5 - CTR + P - TEFb ( + vs . - ATP ) E Fluorescence microscopy images showing the phase - separated droplets formed in 50 mM NaCl containing buffer with 10 μM AFF4 - IDR - eGFP and 10 μM SPT5 - CTR - mCherry or SPT5 - CTR - TE - mCherry in the presence of 10 % PEG - 8000 . F Droplet number ( top ) and droplet area ( bottom ) analyses are shown . Central band is median ; boxes represent 1st and 3rd quartile ( 25th and 75th percentile , respectively ) and whiskers 1 . 5 × interquartile range . Results are representative of two biological replicates , each n > 6 . Two - tailed , unpaired Student ' s t test was performed . AFF4 - IDR + SPT5 - CTR - TE vs . AFF4 - IDR + SPT5 - CTR , * * p = 0 . 0029 .

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 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

Figure 6B Fluorescence microscopy images showing that 2 μM SPT5-CTR-eGFP formed phase-separated droplets in the NaCl containing buffer. C Droplet number (top) and droplet area (bottom) analyses are shown. Central band is median; boxes represent 1st and 3rd quartile (25th and 75th percentile, respectively) and whiskers 1.5× interquartile range. Results are representative of two biological replicates, each n > 6. Two-tailed, unpaired Student's t test was performed. For droplet number, SPT5-CTR + P-TEFb (- ATP) vs. SPT5-CTR, p = **0.0015. SPT5-CTR + P-TEFb (+ vs.- ATP), **p = 0.0032. For droplet area, SPT5-CTR + P-TEFb (+ vs. - ATP)E Fluorescence microscopy images showing the phase-separated droplets formed in 50 mM NaCl containing buffer with 10 μM AFF4-IDR-eGFP and 10 μM SPT5-CTR-mCherry or SPT5-CTR-TE-mCherry in the presence of 10% PEG-8000. F Droplet number (top) and droplet area (bottom) analyses are shown. Central band is median; boxes represent 1st and 3rd quartile (25th and 75th percentile, respectively) and whiskers 1.5× interquartile range. Results are representative of two biological replicates, each n > 6. Two-tailed, unpaired Student's t test was performed. AFF4-IDR + SPT5-CTR-TE vs. AFF4-IDR + SPT5-CTR, **p = 0.0029.


Figure 7A Confocal images showing co - localization of AFF4 with SPT5 in nuclear puncta in AFF4 wild type ( WT ) or R258W cells . B Dotplot ( left ) showing the AFF4 or SPT5 puncta area in AFF4 WT or R258W cells ( Fig 7A ) . Two - tailed , unpaired Student ' s t test was performed . AFF4 R258W vs . WT , * p = 0 . 0254 . Violin plot ( right ) showing the Pearson correlation coefficient of co - localization ratio ( Fig 7A ) . Each n > 20 . Results are representative of three biological replicates . Two - tailed , unpaired Student ' s t test was performed . AFF4 R258W vs . WT , * p = 0 . 0166 . CC Droplet experiments showing that CHOPS syndrome patient missense mutations reinforced AFF4 - ALF domain droplet formation . D Dotplot showing the droplet areas in Fig 7C . Fields per condition n = 5 . Two - tailed , unpaired Student ' s t test was performed . T254S , T254A or R258W vs . WT , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . E AFF4 - ALF - mCherry condensed fraction as a function of AFF4 - ALF - mCherry concentration for experiments in Fig 7C . Error bars represent standard deviations . Fields per condition n = 5 . Two - tailed , unpaired Student ' s t test was performed . T254S , T254A or R258W vs . WT , * p < 0 . 05 , * * p < 0 . 01 . FK - N ChIP - qPCR showing the occupancies of Pol II ( K ) , AFF4 ( L ) , SPT5 ( M ) , and PAF1 ( N ) at the promoter and gene body of HSP70 in AFF4 WT and R258W HEK - 293T cells after heat shock for 2 hours . The HEMO gene serves as a negative control for ChIP - qPCR . Error bars represent standard error of means . Results are plotted with four technical repeats from two biological replicates . Two - tailed , unpaired Student ' s t test was performed . For Pol II ChIP , HSP70 _ promoter , * * * p < 0 . 0001 ; HSP70 _ gene body , * * * p = 0 . 0002 . For AFF4 ChIP , HSP70 _ promoter , * * * p < 0 . 0001 ; HSP70 _ gene body , * * * p < 0 . 0001 . For SPT5 ChIP , HSP70 _ promoter , * * * p < 0 . 0001 ; HSP70 _ gene body , * * p = 0 . 0086 . For PAF1 ChIP , HSP70 _ promoter , * * * p = 0 . 0007 ; HSP70 _ gene body , * * p = 0 . 0021 .

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 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 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

Figure 7A Confocal images showing co-localization of AFF4 with SPT5 in nuclear puncta in AFF4 wild type (WT) or R258W cells. B Dotplot (left) showing the AFF4 or SPT5 puncta area in AFF4 WT or R258W cells (Fig 7A). Two-tailed, unpaired Student's t test was performed. AFF4 R258W vs. WT, *p = 0.0254. Violin plot (right) showing the Pearson correlation coefficient of co-localization ratio (Fig 7A). Each n > 20. Results are representative of three biological replicates. Two-tailed, unpaired Student's t test was performed. AFF4 R258W vs. WT, *p = 0.0166. CC Droplet experiments showing that CHOPS syndrome patient missense mutations reinforced AFF4-ALF domain droplet formation. D Dotplot showing the droplet areas in Fig 7C. Fields per condition n = 5. Two-tailed, unpaired Student's t test was performed. T254S, T254A or R258W vs. WT, *p < 0.05, **p < 0.01, ***p < 0.001. E AFF4-ALF-mCherry condensed fraction as a function of AFF4-ALF-mCherry concentration for experiments in Fig 7C. Error bars represent standard deviations. Fields per condition n = 5. Two-tailed, unpaired Student's t test was performed. T254S, T254A or R258W vs. WT, *p < 0.05, **p < 0.01. FK-N ChIP-qPCR showing the occupancies of Pol II (K), AFF4 (L), SPT5 (M), and PAF1 (N) at the promoter and gene body of HSP70 in AFF4 WT and R258W HEK-293T cells after heat shock for 2 hours. The HEMO gene serves as a negative control for ChIP-qPCR. Error bars represent standard error of means. Results are plotted with four technical repeats from two biological replicates. Two-tailed, unpaired Student's t test was performed. For Pol II ChIP, HSP70_promoter, ***p < 0.0001; HSP70_gene body, ***p = 0.0002. For AFF4 ChIP, HSP70_promoter, ***p < 0.0001; HSP70_gene body, ***p < 0.0001. For SPT5 ChIP, HSP70_promoter, ***p < 0.0001; HSP70_gene body, **p = 0.0086. For PAF1 ChIP, HSP70_promoter, ***p = 0.0007; HSP70_gene body, **p = 0.0021.


Figure 1Western blot analysis and quantitation of Sel1L - Hrd1 ERAD proteins in the livers of WT mice at 3 , 9 and 24 weeks of age ( n = 3 - 6 per group , 2 independent repeats ) . α - tubulin , loading controls for Western blot analysis . Western blot analysis and quantitation of Sel1L - Hrd1 ERAD proteins in the livers of 10 - week - old WT mice under fasted ( overnight ) or ad libitum fed conditions ( n = 3 - 6 per group , 2 independent repeats ) . α - tubulin , loading controls for Western blot analysis . Western blot analysis and quantitation of ERAD proteins in Sel1Lf / f and Sel1LAlb livers ( n = 4 per group , 3 independent repeats ) . Hsp90 loading controls for Western blot analysis . Growth curves of male ( n = 10 each ) and female ( n = 7 each ) mice . Representative images ( E ) and nose - to - anus length ( F ) of male mice at 6 weeks of age ( n = 6 - 10 per group ) . Organ - to - body - weight ratios of liver and kidney in 6 - week - old male mice ( n = 6 per group , 3 independent repeats ) . Daily food intake ( g / d ) normalized to gram of body weight ( gbw ) ( n = 3 per group , measured over 3 days ) . Representative estrus cycle mapping in 2 - 4 - month old females ( n = 6 per group ) . H & E images of paraffin - embedded liver sections from 9 - week - old mice ( n = 4 per group , 3 independent repeats ) . Western blot analysis of cell death ( cleaved and pro - caspase - 3 ) in livers of 9 - week - old mice , with WT ileum as positive control ( n = 3 per group , 3 independent repeats ) . Hsp90 loading controls for Western blot analysis . Representative transmission electron microscope ( TEM ) images obtained from 9 - week - old female mice livers ( n = 10 - 12 cells from one mouse each ) . N , nucleus ; mito , mitochondria ; ER , endoplasmic reticulum .

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Figure 1Western blot analysis and quantitation of Sel1L-Hrd1 ERAD proteins in the livers of WT mice at 3, 9 and 24 weeks of age (n=3-6 per group, 2 independent repeats). α-tubulin, loading controls for Western blot analysis.Western blot analysis and quantitation of Sel1L-Hrd1 ERAD proteins in the livers of 10-week-old WT mice under fasted (overnight) or ad libitum fed conditions (n=3-6 per group, 2 independent repeats). α-tubulin, loading controls for Western blot analysis.Western blot analysis and quantitation of ERAD proteins in Sel1Lf/f and Sel1LAlb livers (n=4 per group, 3 independent repeats). Hsp90 loading controls for Western blot analysis.Growth curves of male (n=10 each) and female (n=7 each) mice.Representative images (E) and nose-to-anus length (F) of male mice at 6 weeks of age (n=6-10 per group).Organ-to-body-weight ratios of liver and kidney in 6-week-old male mice (n=6 per group, 3 independent repeats).Daily food intake (g/d) normalized to gram of body weight (gbw) (n=3 per group, measured over 3 days).Representative estrus cycle mapping in 2-4-month old females (n=6 per group).H&E images of paraffin-embedded liver sections from 9-week-old mice (n=4 per group, 3 independent repeats).Western blot analysis of cell death (cleaved and pro-caspase-3) in livers of 9-week-old mice, with WT ileum as positive control (n=3 per group, 3 independent repeats). Hsp90 loading controls for Western blot analysis.Representative transmission electron microscope (TEM) images obtained from 9-week-old female mice livers (n=10-12 cells from one mouse each). N, nucleus; mito, mitochondria; ER, endoplasmic reticulum.


Figure 2Volcano plot depicting transcriptomics data from the livers of 9 - week - old Sel1Lf / f and Sel1LAlb mice ( n = 3 per group ) ; dotted line marks p = 0 . 05 ; black dots represent fold change > 2 . qPCR analyses of Fgf21 expression in 9 - week - old livers ( n = 3 - 6 per group , 3 independent repeats ) . Ribosomal L32 , loading control for qPCR analysis . Western blot analyses of Fgf21 expression in 9 - week - old livers ( n = 3 - 6 per group , 3 independent repeats ) . α - tubulin , loading control for Western blot analysis . ELISA analysis of Fgf21 in serum from 8 - 9 - week - old mice ( n = 6 - 7 per group ) . Protein levels of Sel1L ( left panel ) and mRNA levels of Fgf21 ( right panel ) in primary mouse hepatocytes isolated from the tamoxifen - inducible Sel1L - knockout Sel1LERCre mice ( 2 independent repeats ) . Hsp90 , loading control for Western blot analysis . Ribosomal L32 , loading control for qPCR analysis . Acute loss - of - function model where 8 - week - old Sel1Lf / f mice were injected i . v . with either AAV8 - Cre or control AAV8 - GFP : Western blot analysis of hepatic and control adipose Sel1L protein ( n = 3 per group ) ; Hsp90 loading control for Western blot analysis . Acute loss - of - function model where 8 - week - old Sel1Lf / f mice were injected i . v . with either AAV8 - Cre or control AAV8 - GFP : qPCR analysis of hepatic Fgf21 expression and ELISA analysis of serum Fgf21 ( n = 3 per group , 2 independent repeats ) . Ribosomal L32 , loading control for qPCR analysis . Heatmaps of top 15 significantly upregulated and downregulated genes in Fgf21 Tg livers and their expression levels in Sel1LAlb livers ( n = 3 per group ) . Scatter plot depicting the logarithmic fold - change ( FC ) for 16 , 402 genes in Sel1LAlb and Fgf21 transgenic ( Tg ) livers ( n = 3 per group ) ; genes that are highly upregulated or downregulated in both datasets are marked in red and blue , respectively ; genes that are upregulated unique to each data set ( e . g . Derl3 for Sel1L - Hrd1 ERAD - deficient liver ) are marked in green . Data from rescue experiments where 5 - week - old Sel1Lf / f and Sel1LAlb mice were injected i . v . with AAV8 - shFgf21 or control AAV8 - shLuc : qPCR analysis of Fgf21 mRNA 3 weeks after injection ( n = 4 per group ) . Ribosomal L32 , loading control for qPCR analysis . Data from rescue experiments where 5 - week - old Sel1Lf / f and Sel1LAlb mice were injected i . v . with AAV8 - shFgf21 or control AAV8 - shLuc : ELISA analysis of Fgf21 in serum ( K ) 3 weeks after injection ( n = 4 per group ) . Data from rescue experiments where 5 - week - old Sel1Lf / f and Sel1LAlb mice were injected i . v . with AAV8 - shFgf21 or control AAV8 - shLuc : Weight gain curve after injection ( n = 7 per group ) .

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Figure 2Volcano plot depicting transcriptomics data from the livers of 9-week-old Sel1Lf/f and Sel1LAlb mice (n=3 per group); dotted line marks p=0.05; black dots represent fold change>2.qPCR analyses of Fgf21 expression in 9-week-old livers (n=3-6 per group, 3 independent repeats). Ribosomal L32, loading control for qPCR analysis.Western blot analyses of Fgf21 expression in 9-week-old livers (n=3-6 per group, 3 independent repeats). α-tubulin, loading control for Western blot analysis.ELISA analysis of Fgf21 in serum from 8-9-week-old mice (n=6-7 per group).Protein levels of Sel1L (left panel) and mRNA levels of Fgf21 (right panel) in primary mouse hepatocytes isolated from the tamoxifen-inducible Sel1L-knockout Sel1LERCre mice (2 independent repeats). Hsp90 , loading control for Western blot analysis. Ribosomal L32, loading control for qPCR analysis.Acute loss-of-function model where 8-week-old Sel1Lf/f mice were injected i.v. with either AAV8-Cre or control AAV8-GFP: Western blot analysis of hepatic and control adipose Sel1L protein (n=3 per group); Hsp90 loading control for Western blot analysis.Acute loss-of-function model where 8-week-old Sel1Lf/f mice were injected i.v. with either AAV8-Cre or control AAV8-GFP: qPCR analysis of hepatic Fgf21 expression and ELISA analysis of serum Fgf21 (n=3 per group, 2 independent repeats). Ribosomal L32, loading control for qPCR analysis.Heatmaps of top 15 significantly upregulated and downregulated genes in Fgf21 Tg livers and their expression levels in Sel1LAlb livers (n=3 per group).Scatter plot depicting the logarithmic fold-change (FC) for 16,402 genes in Sel1LAlb and Fgf21 transgenic (Tg) livers (n=3 per group); genes that are highly upregulated or downregulated in both datasets are marked in red and blue, respectively; genes that are upregulated unique to each data set (e.g. Derl3 for Sel1L-Hrd1 ERAD-deficient liver) are marked in green.Data from rescue experiments where 5-week-old Sel1Lf/f and Sel1LAlb mice were injected i.v. with AAV8-shFgf21 or control AAV8-shLuc: qPCR analysis of Fgf21 mRNA 3 weeks after injection (n=4 per group). Ribosomal L32, loading control for qPCR analysis.Data from rescue experiments where 5-week-old Sel1Lf/f and Sel1LAlb mice were injected i.v. with AAV8-shFgf21 or control AAV8-shLuc: ELISA analysis of Fgf21 in serum (K) 3 weeks after injection (n=4 per group).Data from rescue experiments where 5-week-old Sel1Lf/f and Sel1LAlb mice were injected i.v. with AAV8-shFgf21 or control AAV8-shLuc: Weight gain curve after injection (n=7 per group).


Figure 3Western blot analysis of p - Stat5 in livers of 9 - week - old mice ( n = 3 per group , 3 independent repeats ) , with quantitation of the ratio of p - to total Stat5 shown below the blot . Hsp90 , loading control for Western blot analysis . qPCR analysis of p - Stat5 - associated growth genes in the livers of 9 - week - old mice ( n = 6 per group , 2 independent repeats ) . Ribosomal L32 , loading control for qPCR analysis . Z ambulatory activity of 9 - week - old male mice as measured over 24 hr ( n = 4 males per group ) . Blood glucose levels in 9 - week - old mice after 6 hr fasting ( n = 6 - 10 per group ) . serum insulin levels in 9 - week - old mice after 6 hr fasting ( n = 6 - 10 per group ) . serum triglyceride ( TG ) levels in 9 - week - old mice after 6 hr fasting ( n = 6 - 10 per group ) . serum cholesterol ( CHOL ) levels in 9 - week - old mice after 6 hr fasting ( n = 6 - 10 per group ) . H & E images of inguinal white adipose tissue ( iWAT ) from 8 - week - old mice ( n = 3 per group , 2 independent repeats ) . qPCR analysis of browning - related genes in iWAT ( n = 3 - 6 per group ) . Ribosomal L32 , loading control for qPCR analysis . Western blot analysis of Ucp1 in iWAT of 8 - week - old mice ( n = 3 - 4 per group , 3 independent repeats ) . Hsp90 , loading control for Western blot analysis . Weight gain curve of male mice after 60 % high fat diet ( HFD ) starting at 5 weeks of age ( n = 4 per group , 2 independent repeats ) . Adipose tissue weight normalized to body weight in male mice following 9 weeks of HFD feeding ( n = 4 per group ) .

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Figure 3Western blot analysis of p-Stat5 in livers of 9-week-old mice (n=3 per group, 3 independent repeats), with quantitation of the ratio of p- to total Stat5 shown below the blot. Hsp90, loading control for Western blot analysis.qPCR analysis of p-Stat5-associated growth genes in the livers of 9-week-old mice (n=6 per group, 2 independent repeats). Ribosomal L32, loading control for qPCR analysis.Z ambulatory activity of 9-week-old male mice as measured over 24 hr (n=4 males per group).Blood glucose levels in 9-week-old mice after 6 hr fasting (n=6-10 per group).serum insulin levels in 9-week-old mice after 6 hr fasting (n=6-10 per group).serum triglyceride (TG) levels in 9-week-old mice after 6 hr fasting (n=6-10 per group).serum cholesterol (CHOL) levels in 9-week-old mice after 6 hr fasting (n=6-10 per group).H&E images of inguinal white adipose tissue (iWAT) from 8-week-old mice (n=3 per group, 2 independent repeats).qPCR analysis of browning-related genes in iWAT (n=3-6 per group). Ribosomal L32, loading control for qPCR analysis.Western blot analysis of Ucp1 in iWAT of 8-week-old mice (n=3-4 per group, 3 independent repeats). Hsp90, loading control for Western blot analysis.Weight gain curve of male mice after 60% high fat diet (HFD) starting at 5 weeks of age (n=4 per group, 2 independent repeats).Adipose tissue weight normalized to body weight in male mice following 9 weeks of HFD feeding (n=4 per group).


Figure 4Western blot analysis of Crebh , Crebh - N , Pparα and Xbp1s in whole cell lysates ( A ) and nuclear extracts ( B ) in WT and Sel1LAlb livers ( n = 3 per group , 3 independent repeats ) with quantitation shown in ( C ) . Hsp90 lamin , loading controls for Western blot analysis . qPCR analysis of Crebh , Ppara and Xbp1s in WT and Sel1LAlb livers ( n = 4 - 6 per group , 2 independent repeats ) . Ribosomal L32 , loading control for qPCR analysis . Western blot analysis of Crebh in acute Sel1L loss of function model as described in Figure 2F - G ( n = 3 per group ) . Hsp90 , loading controls for Western blot analysis . Western blot analysis of Crebh protein in 9 - week - old mice ( n = 3 per group , 2 independent repeats ) . WT mice injected i . p . with tunicamycin ( Tm , 1 . 5 µg / g body weight ) for 72 hours were included as a control . Hsp90 loading controls for Western blot analysis . Western blot analysis of Crebh in the livers of 9 - week - old mice after NP40 - detergent fractionation into NP40 soluble ( NP40S ) and pellet ( NP40P ) ( n = 3 per group , 2 independent repeats ) . Hsp90 H2A loading controls for Western blot analysis . Representative confocal images of Crebh in the liver cryosections of 8 - week - old mice ( zoomed out versions in Figure EV3F ) . Note that a fraction of hepatocytes is binucleated . Yellow arrows represent Crebh staining inside in the hepatocyte nucleus . ChIP analysis of Crebh binding onto the Fgf21 promoter in the livers of 9 - week - old mice , normalized first to 5 % input group and then to no - antibody ChIP samples ( n = 3 pooled per group , 2 independent repeats ) . Western blot analysis of SEL1L , HRD1 and CREBH proteins in human Hep3B hepatocytes upon CRISPR deletion of SEL1L with two different guides . , β - actin loading controls for Western blot analysis . qPCR analysis of SEL1L , HRD1 and FGF21 ( K ) in human Hep3B hepatocytes upon CRISPR deletion of SEL1L with two different guides . Ribosomal L32 , loading control for qPCR analysis .

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 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 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 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 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

Figure 4Western blot analysis of Crebh, Crebh-N, Pparα and Xbp1s in whole cell lysates (A) and nuclear extracts (B) in WT and Sel1LAlb livers (n=3 per group, 3 independent repeats) with quantitation shown in (C). Hsp90 lamin, loading controls for Western blot analysis.qPCR analysis of Crebh, Ppara and Xbp1s in WT and Sel1LAlb livers (n=4-6 per group, 2 independent repeats). Ribosomal L32, loading control for qPCR analysis.Western blot analysis of Crebh in acute Sel1L loss of function model as described in Figure 2F-G (n=3 per group). Hsp90 , loading controls for Western blot analysis.Western blot analysis of Crebh protein in 9-week-old mice (n=3 per group, 2 independent repeats). WT mice injected i.p. with tunicamycin (Tm, 1.5 µg/g body weight) for 72 hours were included as a control. Hsp90 loading controls for Western blot analysis.Western blot analysis of Crebh in the livers of 9-week-old mice after NP40-detergent fractionation into NP40 soluble (NP40S) and pellet (NP40P) (n=3 per group, 2 independent repeats). Hsp90 H2A loading controls for Western blot analysis.Representative confocal images of Crebh in the liver cryosections of 8-week-old mice (zoomed out versions in Figure EV3F). Note that a fraction of hepatocytes is binucleated. Yellow arrows represent Crebh staining inside in the hepatocyte nucleus.ChIP analysis of Crebh binding onto the Fgf21 promoter in the livers of 9-week-old mice, normalized first to 5% input group and then to no-antibody ChIP samples (n=3 pooled per group, 2 independent repeats).Western blot analysis of SEL1L, HRD1 and CREBH proteins in human Hep3B hepatocytes upon CRISPR deletion of SEL1L with two different guides. , β-actin loading controls for Western blot analysis.qPCR analysis of SEL1L, HRD1 and FGF21 (K) in human Hep3B hepatocytes upon CRISPR deletion of SEL1L with two different guides. Ribosomal L32, loading control for qPCR analysis.


Figure 5Western blot analysis of Crebh protein half - life in transfected WT and HRD1 - / - HEK293T cells treated with cycloheximide ( CHX ) for indicated times . The decay of protein from one experiment is shown below . Hsp90 , loading control for Western blot analysis . Western blot analysis and quantitation of Crebh in Crebh - transfected WT ( B ) and HRD1 - / - ( C ) HEK293T cells pre - treated with the proteasomal inhibitor bortezomib ( BTZ ) or lysosomal inhibitor chloroquine ( CHQ ) for 2 hours and then with CHX for additional 1 hour ( n = 2 per group , 2 independent repeats ) . Hsp90 , loading control for Western blot analysis . Western blot analysis of Crebh ubiquitination following immunoprecipitation ( IP ) of Crebh - Flag and Crebh - N - Flag in HEK293T cells transfected with indicated plasmids . Samples were boiled with SDS before IP for denaturing IP and not so for native IP . These cells were treated with proteasomal inhibitor BTZ for the last 6 hr prior to immunoprecipitation . Hsp90 , loading control for Western blot analysis . Western blot analysis of endoglycosidase H ( endoH ) - sensitivity of Crebh in the livers of 9 - week - old mice ; ' r ' refers to endoH - cleavage - resistant species and ' s ' refers to endoH - cleavage - sensitive species ( n = 3 per group , 2 independent repeats ) . Hsp90 , loading control for Western blot analysis .

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 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 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

Figure 5Western blot analysis of Crebh protein half-life in transfected WT and HRD1-/- HEK293T cells treated with cycloheximide (CHX) for indicated times. The decay of protein from one experiment is shown below. Hsp90, loading control for Western blot analysis.Western blot analysis and quantitation of Crebh in Crebh-transfected WT (B) and HRD1-/- (C) HEK293T cells pre-treated with the proteasomal inhibitor bortezomib (BTZ) or lysosomal inhibitor chloroquine (CHQ) for 2 hours and then with CHX for additional 1 hour (n=2 per group, 2 independent repeats). Hsp90, loading control for Western blot analysis.Western blot analysis of Crebh ubiquitination following immunoprecipitation (IP) of Crebh-Flag and Crebh-N-Flag in HEK293T cells transfected with indicated plasmids. Samples were boiled with SDS before IP for denaturing IP and not so for native IP. These cells were treated with proteasomal inhibitor BTZ for the last 6 hr prior to immunoprecipitation. Hsp90, loading control for Western blot analysis.Western blot analysis of endoglycosidase H (endoH)-sensitivity of Crebh in the livers of 9-week-old mice; 'r' refers to endoH-cleavage-resistant species and 's' refers to endoH-cleavage-sensitive species (n=3 per group, 2 independent repeats). Hsp90, loading control for Western blot analysis.


Figure 65 - week - old Sel1Lf / f and Sel1LAlb mice were injected i . v . once with AAV8 - shCrebh or control AAV8 - shLuc . Western blot analysis of hepatic Sel1L and Crebh 5 weeks post injection ( n = 3 mice each , 2 independent repeats ) . Hsp90 , loading control for Western blot analysis . 5 - week - old Sel1Lf / f and Sel1LAlb mice were injected i . v . once with AAV8 - shCrebh or control AAV8 - shLuc . qPCR analysis of Crebh and Fgf21 mRNA 5 weeks post injection ( n = 3 per group , 2 independent repeats ) . Ribosomal L32 , loading control for qPCR analysis . 5 - week - old Sel1Lf / f and Sel1LAlb mice were injected i . v . once with AAV8 - shCrebh or control AAV8 - shLuc . ELISA analysis of circulating Fgf21 ( C ) 5 weeks post injection ( n = 3 per group , 2 independent repeats ) . 5 - week - old Sel1Lf / f and Sel1LAlb mice were injected i . v . once with AAV8 - shCrebh or control AAV8 - shLuc . qPCR analysis of hepatic growth - associated genes 5 weeks after injection ( n = 6 per group ) . Ribosomal L32 , loading control for qPCR analysis . 5 - week - old Sel1Lf / f and Sel1LAlb mice were injected i . v . once with AAV8 - shCrebh or control AAV8 - shLuc . Weight gain 6 weeks post injection ( n = 10 per group ) . 5 - week - old Sel1Lf / f and Sel1LAlb mice were injected i . v . once with AAV8 - shCrebh or control AAV8 - shLuc . Insulin tolerance test ( ITT ) 5 - weeks after injection ( n = 10 per group ) . 5 - week - old Sel1Lf / f and Sel1LAlb mice were injected i . v . once with AAV8 - shCrebh or control AAV8 - shLuc . qPCR of Ucp1 levels in inguinal white adipose tissue ( iWAT ) 5 weeks post injection ( n = 3 per group , 2 independent repeats ) . Ribosomal L32 , loading control for qPCR analysis . 5 - week - old Sel1Lf / f and Sel1LAlb mice were injected i . v . once with AAV8 - shCrebh or control AAV8 - shLuc . Western blot analysis ( H ) of Ucp1 levels in inguinal white adipose tissue ( iWAT ) 5 weeks post injection ( n = 3 per group , 2 independent repeats ) . Hsp90 , loading control for Western blot analysis .

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 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 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 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 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 65-week-old Sel1Lf/f and Sel1LAlb mice were injected i.v. once with AAV8-shCrebh or control AAV8-shLuc. Western blot analysis of hepatic Sel1L and Crebh 5 weeks post injection (n=3 mice each, 2 independent repeats). Hsp90, loading control for Western blot analysis.5-week-old Sel1Lf/f and Sel1LAlb mice were injected i.v. once with AAV8-shCrebh or control AAV8-shLuc. qPCR analysis of Crebh and Fgf21 mRNA 5 weeks post injection (n=3 per group, 2 independent repeats). Ribosomal L32, loading control for qPCR analysis.5-week-old Sel1Lf/f and Sel1LAlb mice were injected i.v. once with AAV8-shCrebh or control AAV8-shLuc. ELISA analysis of circulating Fgf21 (C) 5 weeks post injection (n=3 per group, 2 independent repeats).5-week-old Sel1Lf/f and Sel1LAlb mice were injected i.v. once with AAV8-shCrebh or control AAV8-shLuc. qPCR analysis of hepatic growth-associated genes 5 weeks after injection (n=6 per group). Ribosomal L32, loading control for qPCR analysis.5-week-old Sel1Lf/f and Sel1LAlb mice were injected i.v. once with AAV8-shCrebh or control AAV8-shLuc. Weight gain 6 weeks post injection (n=10 per group).5-week-old Sel1Lf/f and Sel1LAlb mice were injected i.v. once with AAV8-shCrebh or control AAV8-shLuc. Insulin tolerance test (ITT) 5-weeks after injection (n=10 per group).5-week-old Sel1Lf/f and Sel1LAlb mice were injected i.v. once with AAV8-shCrebh or control AAV8-shLuc. qPCR of Ucp1 levels in inguinal white adipose tissue (iWAT) 5 weeks post injection (n=3 per group, 2 independent repeats). Ribosomal L32, loading control for qPCR analysis.5-week-old Sel1Lf/f and Sel1LAlb mice were injected i.v. once with AAV8-shCrebh or control AAV8-shLuc. Western blot analysis (H) of Ucp1 levels in inguinal white adipose tissue (iWAT) 5 weeks post injection (n=3 per group, 2 independent repeats). Hsp90, loading control for Western blot analysis.


Figure 7Analysis of correlation between Sel1L - Hrd1 ERAD and Crebh - Fgf21 during fasting - feeding Western blot analysis of hepatic Sel1L - Hrd1 ERAD and Crebh of nuclear ( Nuc ) and cytosolic ( Cyto ) fractions from 10 - week - old Sel1Lf / f and Sel1LAlb mice under overnight fasted or fed states . Analysis of correlation between Sel1L - Hrd1 ERAD and Crebh - Fgf21 during fasting - feeding Quantitation of levels of Sel1L / Hrd1 / Crebh proteins and Fgf21 mRNA in the livers of 10 - week - old Sel1Lf / f mice . during fasting - feeding Quantitation of total Crebh ( Crebh + Crebh - N ) protein levels in the livers of 10 - week - old Sel1Lf / f and Sel1LAlb mice . during fasting - feeding Serum Fgf21 levels in 10 - week - old Sel1Lf / f and Sel1LAlb mice . Analysis of correlation between Sel1L - Hrd1 ERAD and Crebh - Fgf21 during growth Western blot analysis of hepatic Sel1L - Hrd1 ERAD and Crebh of nuclear ( Nuc ) and cytosolic ( Cyto ) fractions from the livers from Sel1Lf / f and Sel1LAlb mice at 3 , 9 and 24 weeks of age . Analysis of correlation between Sel1L - Hrd1 ERAD and Crebh - Fgf21 during growth Quantitation of levels of Sel1L / Hrd1 / Crebh proteins and Fgf21 mRNA with the livers from Sel1Lf / f and Sel1LAlb mice at 3 , 9 and 24 weeks of age . Quantitation of total Crebh ( Crebh + Crebh - N ) protein levels in the livers from Sel1Lf / f and Sel1LAlb mice at 3 , 9 and 24 weeks of age . Serum Fgf21 levels in the livers from Sel1Lf / f and Sel1LAlb mice at 3 , 9 and 24 weeks of age .

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Figure 7Analysis of correlation between Sel1L-Hrd1 ERAD and Crebh-Fgf21 during fasting-feeding Western blot analysis of hepatic Sel1L-Hrd1 ERAD and Crebh of nuclear (Nuc) and cytosolic (Cyto) fractions from 10-week-old Sel1Lf/f and Sel1LAlb mice under overnight fasted or fed states.Analysis of correlation between Sel1L-Hrd1 ERAD and Crebh-Fgf21 during fasting-feeding Quantitation of levels of Sel1L/Hrd1/Crebh proteins and Fgf21 mRNA in the livers of 10-week-old Sel1Lf/f mice.during fasting-feeding Quantitation of total Crebh (Crebh+Crebh-N) protein levels in the livers of 10-week-old Sel1Lf/f and Sel1LAlb mice.during fasting-feeding Serum Fgf21 levels in 10-week-old Sel1Lf/f and Sel1LAlb mice.Analysis of correlation between Sel1L-Hrd1 ERAD and Crebh-Fgf21 during growth Western blot analysis of hepatic Sel1L-Hrd1 ERAD and Crebh of nuclear (Nuc) and cytosolic (Cyto) fractions from the livers from Sel1Lf/f and Sel1LAlb mice at 3, 9 and 24 weeks of age.Analysis of correlation between Sel1L-Hrd1 ERAD and Crebh-Fgf21 during growth Quantitation of levels of Sel1L/Hrd1/Crebh proteins and Fgf21 mRNA with the livers from Sel1Lf/f and Sel1LAlb mice at 3, 9 and 24 weeks of age.Quantitation of total Crebh (Crebh+Crebh-N) protein levels in the livers from Sel1Lf/f and Sel1LAlb mice at 3, 9 and 24 weeks of age.Serum Fgf21 levels in the livers from Sel1Lf/f and Sel1LAlb mice at 3, 9 and 24 weeks of age.