D Heat map showing log2 fold change differences of interferon - related genes expression between ENU treatment control ( saline ) in WT G1 Tert - / - mice by - seq . n = 3 mice per group . E Heat map showing log2 fold change differences of cytokine / chemokine between ENU treatment control ( saline ) in WT G1 Tert - / - mice by cytokine / chemokine array . n = 5 mice per group .

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D Heat map showing log2 fold change differences of interferon-related genes expression between ENU treatment and control (saline) in WT and G1 Tert-/- mice by RNA-seq. n = 3 mice per group. E Heat map showing log2 fold change differences of cytokine/chemokine between ENU treatment and control (saline) in WT and G1 Tert-/- mice by cytokine/chemokine array. n = 5 mice per group. F


B TCGA fragments per kilobase million ( FPKM ) values of TERT , CXCL10 , ISG15 , OASL in tumours grouped in TERThigh ( n = 500 ) TERTlow groups ( n = 500 ) . Data represent mean ± SD . P - values were analysed using unpaired two - Student ' s t - .

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B TCGA fragments per kilobase million (FPKM) values of TERT, CXCL10, ISG15, and OASL in tumours grouped in TERThigh (n = 500) and TERTlow groups (n = 500). Data represent mean ± SD. P-values were analysed using unpaired two-tailed Student's t-tests.


C TCGA FPKM values of interferon - related genes in tumours grouped in TERThigh ( n = 500 ) TERTlow ( n = 500 ) groups . Heat map showing mean FPKM values of each gene .

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C TCGA FPKM values of interferon-related genes in tumours grouped in TERThigh (n = 500) and TERTlow (n = 500) groups. Heat map showing mean FPKM values of each gene.


Expression of TERT ( D ) in TERThigh TERTlow cancer types ( 6 types in each ) . ACC ( adrenocortical carcinoma ) , n = 79 . KICH ( kidney chromophobe ) , n = 66 . KIRP ( kidney renal papillary carcinoma ) , n = 291 . LGG ( brain lower grade glioma ) , n = 532 . PRAD , n = 502 . THCA , n = 513 . BLCA ( bladder urothelial carcinoma ) , n = 414 . CESC ( cervical squamous carcinoma endocervical adenocarcinoma ) , n = 306 . DLBC ( lymphoid neoplasm diffuse large B - lymphoma ) , n = 48 . LUSC ( lung squamous carcinoma ) , n = 502 . OV ( ovarian serous cystadenocarcinoma ) , n = 430 . boxplots include median , box indicates interquartile range ( IQR ) whiskers denote 1 . 5 × IQR . P - values were analysed using two - Mann - Whitney U .

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Expression of TERT (D) in TERThigh and TERTlow cancer types (6 types in each). ACC (adrenocortical carcinoma), n = 79. KICH (kidney chromophobe), n = 66. KIRP (kidney renal papillary cell carcinoma), n = 291. LGG (brain lower grade glioma), n = 532. PRAD, n = 502. THCA, n = 513. BLCA (bladder urothelial carcinoma), n = 414. CESC (cervical squamous cell carcinoma and endocervical adenocarcinoma), n = 306. DLBC (lymphoid neoplasm diffuse large B-cell lymphoma), n = 48. LUSC (lung squamous cell carcinoma), n = 502. OV (ovarian serous cystadenocarcinoma), n = 430. All boxplots include the median line, box indicates the interquartile range (IQR) and whiskers denote the 1.5 × IQR. P-values were analysed using two-tailed Mann-Whitney U test.


Expression of DDX58 , IFIH1 , CXCL10 , IFI44 , ISG15 , OASL ( E ) in TERThigh TERTlow cancer types ( 6 types in each ) . ACC ( adrenocortical carcinoma ) , n = 79 . KICH ( kidney chromophobe ) , n = 66 . KIRP ( kidney renal papillary carcinoma ) , n = 291 . LGG ( brain lower grade glioma ) , n = 532 . PRAD , n = 502 . THCA , n = 513 . BLCA ( bladder urothelial carcinoma ) , n = 414 . CESC ( cervical squamous carcinoma endocervical adenocarcinoma ) , n = 306 . DLBC ( lymphoid neoplasm diffuse large B - lymphoma ) , n = 48 . LUSC ( lung squamous carcinoma ) , n = 502 . OV ( ovarian serous cystadenocarcinoma ) , n = 430 . boxplots include median , box indicates interquartile range ( IQR ) whiskers denote 1 . 5 × IQR . P - values were analysed using two - Mann - Whitney U .

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Expression of DDX58, IFIH1, CXCL10, IFI44, ISG15, and OASL (E) in TERThigh and TERTlow cancer types (6 types in each). ACC (adrenocortical carcinoma), n = 79. KICH (kidney chromophobe), n = 66. KIRP (kidney renal papillary cell carcinoma), n = 291. LGG (brain lower grade glioma), n = 532. PRAD, n = 502. THCA, n = 513. BLCA (bladder urothelial carcinoma), n = 414. CESC (cervical squamous cell carcinoma and endocervical adenocarcinoma), n = 306. DLBC (lymphoid neoplasm diffuse large B-cell lymphoma), n = 48. LUSC (lung squamous cell carcinoma), n = 502. OV (ovarian serous cystadenocarcinoma), n = 430. All boxplots include the median line, box indicates the interquartile range (IQR) and whiskers denote the 1.5 × IQR. P-values were analysed using two-tailed Mann-Whitney U test.


G Representative sections of immunohistochemical of CD4 FOXP3 in TERT - ERVshigh TERT - ERVslow colon tumours . Red arrowheads indicate FOXP3 + . Scale bar = 100 μm .

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G Representative sections of immunohistochemical staining of CD4 and FOXP3 in TERT-ERVshigh and TERT-ERVslow colon tumours. Red arrowheads indicate FOXP3+ cells. Scale bar = 100 μm.


Chaining indices of indicated genotypes . Statistical significance was compared with control . ; n = 9 , 6 , 7 ( E ) groups ( 10 flies per group ) . Courtship indices for one target ( Canton - S ) one tester of indicated genotypes . Changes were determined by comparing data with control . ; n = 9 , 8 , 9 ( F ) flies . Sexual preference of single of indicated genotypes toward a decapitated virgin a decapitated naïve . Statistical significance was compared with other gender . ; n = 10 , 10 , 10 ( F ) flies .

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Chaining indices of the indicated genotypes. Statistical significance was compared with control. ; n = 9, 6, 7 (E) groups (10 flies per group). Courtship indices for one target (Canton-S) male and one tester male of the indicated genotypes. Changes were determined by comparing the data with control. ; n = 9, 8, 9 (F) flies. Sexual preference of single male of indicated genotypes toward a decapitated virgin female and a decapitated naïve male. Statistical significance was compared with the other gender. ; n = 10, 10, 10 (F) flies.


Courtship indices for one target ( Canton - S ) one tester of indicated genotypes . Changes were determined by comparing data with relative control . n = 8 , 13 , 13 ( D )

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Courtship indices for one target (Canton-S) male and one tester male of the indicated genotypes. Changes were determined by comparing the data with relative control. n = 8, 13, 13 (D)


( A - C ) DA level ( pictograms per head ) in of indicated genotypes . n = 3 , 3 , 3 ( A ) ; n = 4 , 5 , 3 , 3 ( B ) ; n = 3 , 3 , 3 ( C ) times ( 100 heads per genotype ) .

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(A-C) DA level (pictograms per head) in males of the indicated genotypes. n = 3, 3, 3 (A); n = 4, 5, 3, 3 (B); n = 3, 3, 3 (C) times (100 heads per genotype).


( D ) Posterior confocal sections of UAS - mCD8 - GFP ; Ddc - Gal4 adult brain in combination with UAS - LacZ or UAS - Myc . RNAi - 1 stained with antibody to nc82 ( pan - neuropil marker ; magenta ) . Scale bars represent 50 μm .

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(D) Posterior confocal sections of UAS-mCD8-GFP; Ddc-Gal4 adult male brain in combination with UAS-LacZ or UAS-Myc.RNAi-1 stained with antibody to nc82 (pan-neuropil marker; magenta). Scale bars represent 50 μm.


( F , G ) Western blot of extracted from adult heads of indicated genotypes . Statistical showing level of GFP ( F ) or Ddc ( G ) to that of β - actin in Western blot .

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(F, G) Western blot of proteins extracted from adult heads of indicated genotypes. Statistical analysis showing the protein level of GFP (F) or Ddc (G) to that of β-actin in Western blot.


( B ) Drosophila S2 transfected with pUAST - HA empty vector or pUAST - HA - Myc plasmid were co - transfected with each of firefly luciferase reporter vector bearing fragment shown in ( A ) . After 48 hours , luciferase activity was detected in each lysate ( n = 5 times ) .

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(B) Drosophila S2 cells transfected with the pUAST-HA empty vector or the pUAST-HA-Myc plasmid were co-transfected with each of the firefly luciferase reporter vector bearing the fragment shown in (A). After 48 hours, the luciferase activity was detected in each cell lysate (n = 5 times).


( C ) Drosophila S2 transfected with pUAST - HA - Myc plasmid or empty vector were used for of ChIP - PCRs ( n = 4 times ) .

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(C) Drosophila S2 cells transfected with the pUAST-HA-Myc plasmid or the empty vector were used for quantification of ChIP-PCRs (n = 4 times).


( D ) from adult heads ( n = 50 heads per genotype ) of indicated genotypes was subjected to real - time qPCR .

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(D) RNA from adult male heads (n = 50 heads per genotype) of indicated genotypes was subjected to real-time qPCR.


( E ) DA level ( pictograms per head ) in head of of indicated genotypes . n = 4 ( 100 heads per genotype ) .

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(E) DA level (pictograms per head) in the head of males of the indicated genotypes. n = 4 (100 heads per genotype).


B . Deconvolved of Chm7 - GFP truncations with Nup170 - mCherry NE marker merge of green red channels . Scale bar is 5 μm .

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B. Deconvolved fluorescence micrographs of Chm7-GFP and truncations with Nup170-mCherry NE marker and merge of green and red channels. Scale bar is 5 μm.


C . Scatter plot of mean with SD of NE foci ( horizontal is mean ) . > 50 foci were measured for each GFP fusion ; p values from un - paired student ' s T - . * * * is p ≤ 0 . 001 ; * * * * is p ≤ 0 . 0001 .

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C. Scatter plot of the mean fluorescence intensity with SD of individual NE foci (horizontal line is mean). >50 foci were measured for each GFP fusion; p values from un-paired student's T-test. *** is p ≤ 0.001; **** is p ≤ 0.0001.


D . Deconvolved of Chm7 - GFP Mps3 - mCherry with merge of green red ( right ) . Scale bar is 5 μm .

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D. Deconvolved fluorescence micrographs of Chm7-GFP and Mps3-mCherry with merge of green and red images (right). Scale bar is 5 μm.


E . 10 - fold serial dilutions of chm7Δapq12Δ strains grown at indicated temperatures with either an empty pRS416 plasmid ( - ) or those expressing indicated Chm7 constructs ( pRS416 - HA - CHM7 , pRS416 - HA - chm7 - NTD , pRS416 - HA - chm7 - CTD ) . See Appendix Figure S2A for .

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E. 10-fold serial dilutions of chm7Δapq12Δ strains grown at the indicated temperatures with either an empty pRS416 plasmid (-) or those expressing the indicated Chm7 constructs (pRS416-HA-CHM7, pRS416-HA-chm7-NTD, pRS416-HA-chm7-CTD). See Appendix Figure S2A for protein levels.


A . Deconvolved inverted Chm7 - GFP truncations in indicated null strains . Scale bar is 5 μm . B . Plot of of with SD of Chm7 - GFP ( truncations ) NE foci from A . Data are from 3 independent replicates where > 200 were counted for each . p values from un - paired student ' s T - . * * is a p value ≤ 0 . 01 ; * * * * , p ≤ 0 . 0001 .

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A. 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 of Chm7 - GFP in a heh1Δ containing either an empty pRS426 plasmid ( - ) or those expressing HEH1 ( pRS426 - HEH1 ) or HEH2 ( pRS426 - HEH2 ) . D . Plots of of from C with Chm7 - GFP NE - foci . Data are from 3 independent replicates where > 200 were counted for each . Error bars are SD . p values from T - . * * , p ≤ 0 . 01 ; * * * * , p ≤ 0 . 0001 .

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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.


C . GST , GST - Snf7 GST - snf7OPEN were immobilized on GT - resin incubated with buffer ( - ) or recombinant heh2 ( 1 - 308 ) - His6 . Bound separated by SDS - PAGE were visualized by coomassie stain by Western blot with anti - His6 antibody 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 ( MW ) markers .

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C. 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 detection of bound by autoradiography ( top panel ) or coomassie ( bottom ) .

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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 .

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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.


A . Deconvolved inverted of BiFC signal of Chm7 - VC with either Heh1 - VN or Heh2 - VN . borders are outlined . Scale bar is 5 μm .

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A. 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 of yeast strains expressing Heh1 - VN Snf7 - VC with either Chm7 - mCherry or Mps3 - mCherry merge of both channels . Scale bar is 5 μm .

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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 of BiFC signal of indicated VN VC fusions in indicated strains . borders outlined . 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 of with BiFC signal of indicated VN VC pairs in indicated null backgrounds . Data are from three independent replicates where > 225 per per replicate were quantified . Error bars are SD from mean of each replicate . p values from un - paired student ' s T - where ns is p > 0 . 05 . * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 ; * * * * , p ≤ 0 . 0001 .

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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 of Heh1 - VN Snf7 - VC BiFC signal in a chm7Δ with either an empty plasmid ( " - " ; pRS416 ) or one expressing CHM7 ( pRS416 - HA - CHM7 ) . borders outlined . Scale bar is 5 μm . G . Plot of of with BiFC signal from F . Error bars are SD from mean of each replicate . p values from un - paired student ' s T - where ns is p > 0 . 05 . * * * , p ≤ 0 . 001 .

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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.


A . Deconvolved inverted of Chm7 - GFP in indicated strains . Scale bar is 5 μm . of ± SD with Chm7 - GFP foci is indicated below each panel .

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A. 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 of SINC - containing nuclei in vps4Δ vps4Δpom152Δ expressing Chm7 - GFP Nup170 - mCherry ( green , red merged are shown ) . Scale bar is 1 μm .

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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 proportion of nup clusters with Chm7 - GFP enrichment in indicated genetic backgrounds . Error bars are SD of mean from 3 independent replicates .

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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 micrograph showing lack of colocalization of Chm7 - GFP clustered Nup170 - mCherry in a nup133Δ . Scale bar is 1 μm .

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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 total / integrated density ( in arbitrary units ( A . U . ) ) of Chm7 - GFP within ( outside of ) SINC . Error bars represent SD deviation of mean from > 50 SINC or non - SINC accumulations pooled from 3 independent replicates . p values from un - paired student ' s T - with Welch ' s correction . * * * * is p ≤ 0 . 0001 .

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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 total / integrated density of SINC - enriched Chm7 - GFP Nup170 - mCherry within SINCs . Linear regression calculated from 150 SINCs pooled from 3 independent replicates ; r is linear correlation ( Pearson ' s ) coefficient .

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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.


A . 10 - fold serial dilutions of indicated yeast strains were grown on YPD at 23 ° C for 3 days before imaging .

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A.10-fold serial dilutions of the indicated yeast strains were grown on YPD at 23°C for 3 days before imaging.


C . Deconvolved of Sna3 - mCherry in indicated genetic backgrounds . Scale bar is 5 μm .

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C. Deconvolved fluorescence micrographs of Sna3-mCherry in the indicated genetic backgrounds. Scale bar is 5 μm.


D . Deconvolved ( maximum projections of a z - series of ) of GFP - Nup49 in indicated yeast strains ( arrows indicate SINCs ) . E . Plot of proportion of in indicated strains with SINCs . Error bars are SD from mean from 3 independent replicates quantifying > 300 for each . p values from un - paired student ' s T - where ns is p > 0 . 05 ; * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 .

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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.


A . Deconvolved inverted of indicated strains expressing Chm7 - GFP ( percentages ± SD of proportion of with Chm7 - GFP foci is at bottom of each panel , see Figure EV5B ) . Scale bar is 5 μm .

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A. 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 of with indicated number of Chm7 - GFP foci . Error bars are SD from mean from 3 independent replicates of > 50 foci per . p values from 2 - way ANOVA where ns represents p > 0 . 05 ; * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ; * * * * , p ≤ 0 . 0001 .

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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 proportion of with Chm7 - GFP NE foci are shown under each panel ( see Figure EV5F ) . D . Plots of of from C with indicated number of Chm7 - GFP foci at 23 ° C ( left ) 37 ° C ( right ) . Error bars are SD from mean from 3 independent replicates of > 50 foci per . p values from 2 - way ANOVA where ns is p > 0 . 05 ; * , p ≤ 0 . 05 ; * * * , p ≤ 0 . 001 .

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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.


A . Plot of of nup116Δ incubated for 3 h at 37 ° C with rim - like accumulations of Chm7 - GFP . Error bars are SD from mean from 3 independent replicates of > 100 . p values from unpaired student ' s T - where * * * is p ≤ 0 . 001 . B . Deconvolved inverted of Chm7 - GFP in nup116Δ at either 23 ° C or grown for 3 h at 37 ° C . Percentages ± SD of proportion of with Chm7 - GFP NE foci are shown under each panel ( see Figure EV5F ) .

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A. 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 showing herniations of NE in nup116Δ after incubation for 3 h at 37 ° C . N denotes interior . Scale bars are 100 nm .

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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 retention of a URA3 / NUP116 plasmid . acquired after incubation for 3 days ( left ) or 6 days ( right ) at 23 ° C .

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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.


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

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A. 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.


( B ) High magnification view of boxed regions in ( A ) pseudo - colored to show actin depletion in anaphase . Scale bar - 1 µm .

(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 of representative Hela expressing mNeon - Green Aurora B Mask to label , exiting mitosis , showing dynamic re - localization of Aurora B from to overlapping microtubules cleavage furrow with control siRNA treatment ( arrows top ) . With knock - down of RACGAP1 , Aurora B still re - localises from to microtubules furrow in midzone in early anaphase ( arrows bottom ) , but microtubule localization is lost at later stages ( asterisk bottom ) . Scale bar - 10 µm .

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(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.


( E ) Stills of representative Hela expressing Lifeact - GFP H2B - mCherry at 10 mins following forced mitotic exit with siControl , siRACGAP1 , DMSO 2µM ZM447439 treatment . Control siRNA , RACGAP1 siRNA DMSO treated show clearance of actin from cortex close to , while ZM447439 treated fail to clear actin , quantified in ( F ) . Note , failure in actin accumulation in RACGAP1 depleted , asterisk . Scale bar - 10

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(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 ) of actin across perimeter of on from , with 5 6 being closest to 1 10 furthest away , as in cartoon ( B ) . Graph shows decrease in actin closer to in siControl ( n = 26 ) , siRACGAP1 ( n = 37 ) Control DMSO treated ( n = 38 ) , seen by lower than 1 ( dotted red ) , while this decrease is absent in ZM447439 treated ( n = 42 ) . Data presented as mean ± SD .

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(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.


( B ) Stills from time - lapse of basal most region of Hela expressing Lifeact - GFP H2B - mCherry forced to undergo flat monopolar mitotic exit . While actin is cleared underneath in control DMSO treated , it fails to be cleared around when are treated with Aurora B inhibitor ZM447439 during mitotic exit . Dotted mask shows position of in actin channel . Scale bar - 10µm .

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(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 ) of clearance of actin underneath for still shown in ( B ) . While under control starts clearing actin within 10 mins , Aurora B inhibited fail to do so .

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(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.


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

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(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.


( C ) of actin at poles of at 6 mins post anaphase onset , before furrow formation under different conditions as in ( A ) . Actin fails to be cleared from poles upon Aurora B kinase inhibition during mitotic exit is stronger when both centralspindlin 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 ZM vs siRACGAP1 + ZM - p = 0 . 1528 .

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(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 ) of shortest between centroid of cortical actomyosin network . While is able to reach close to cortex in both siControl siRACGAP1 treated , of from cortex increases upon ZM treatment is further enhanced in 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 .

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(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 .


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

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

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


A ) shows log2FC alterations between significantly different inflammation related on Day 70 vs Day 0 in CMA placebo groups . Asterisks indicate statistical significance on paired Student ' s t . p < 0 . 05 ;

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A) 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 ) shows correlation between plasma of inflammation related plasma of metabolic activators including serine , cysteine , carnitine nicotinamide . Asterisks indicate statistical significance on Spearman correlation . p < 0 . 05 ; shows associations between significantly different inflammation related ( CD8A , CSF - 1 , CCL23 , FGF - 21 OSM )

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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 on Spearman correlation . p < 0 . 05 ; shows associations between significantly different inflammation related ( CD8A , CSF - 1 , CCL23 , FGF - 21 OSM ) C ) with 10 most significantly correlated plasma metabolites ,

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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,


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

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Scatter 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).


shows association between plasma level of clinical variables including liver fat , ALT , AST , uric acid , creatinine with , B ) plasma level of 10 most significant inflammation related

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

Heatmap 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 proteins


shows association between plasma level of clinical variables including liver fat , ALT , AST , uric acid , creatinine with , C ) abundance of species in gut

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

Heatmap 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 microbiome


shows association between plasma level of clinical variables including liver fat , ALT , AST , uric acid , creatinine with D ) abundance of species in oral microbiome . AST , Aspartate aminotransferase ; ALT , Alanine transaminase . Asterisks indicate statistical significance on Spearman correlation . p < 0 . 05 ; Cor . Coeff : Correlation coefficient .

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Heatmap 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.


A , B ( A ) Confocal imaging of FOS FISH with concurrent SPT5 Pol II - S5P IF showing that SPT5 Pol II - S5P occupied FOS loci after serum starvation . Zoomed - in views of merged regions are indicated by arrow . ( B ) Same as Fig 1A . but for FOS FISH with concurrent SPT5 NELFE IF .

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A, 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 showing that SPT5 , NELFA , Pol II - S5P AFF4 formed in HCT 116 before after treated with 1 % 1 , 6 - hexanediol ( abbreviation as 1 , 6 - hex ) for 30 minutes . F Violin plot showing number of containing in Fig 1E . Results are representative of three biological replicates , each n > 20 . Two - , unpaired Student ' s t was performed . For SPT5 , p = 0 . 1301 . For NELFA , p = 0 . 2756 . For Pol II - S5P , p = 0 . 8179 . For AFF4 , * * * p < 0 . 0001 .

O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O

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. G


G - I ChIP - qPCR showing that occupancies of Pol II ( G ) , SPT5 ( H ) , NELFE ( I ) at promoters gene bodies of HSP70 , GAPDH , MYC in HeLa . 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 - , unpaired Student ' s t was performed . For Pol II ChIP , HSP70 _ promoter , * * p = 0 . 0014 ; GAPDH _ promoter , p = 0 . 8385 ; GAPDH _ gene , * * * p < 0 . 0001 ; MYC _ promoter , p = 0 . 1765 ; MYC _ gene , * * * p = 0 . 0002 . For SPT5 ChIP , HSP70 _ promoter , * * p = 0 . 0033 ; GAPDH _ promoter , p = 0 . 9603 ; GAPDH _ gene , * * p = 0 . 0012 ; MYC _ promoter , p = 0 . 9898 ; MYC _ gene , * * * p < 0 . 0001 . For NELFE ChIP , HSP70 _ promoter , p = 0 . 3913 ; GAPDH _ promoter , p = 0 . 9347 ; MYC _ promoter , p = 0 . 8558 .

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G-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.


C Normalized FRAP curves for eGFP - SPT5 . bleaching events occurred at 0 second . Confocal were embedded within plot showing FRAP of in vitro eGFP - SPT5 clusters . Results shown are from three biological replicates . arrowhead represents bleached region .

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C 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 imaging of HEK - 293T expressing eGFP - SPT5 together with mCherry - SPT5 , mCherry - SPT5ΔNTR , or mCherry - SPT5ΔCTR close to endogenous level . F Violin plot showing clusters ( top ) 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 - , unpaired Student ' s t was performed . * * * p < 0 . 001 .

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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. G


H Confocal showing FRAP of NELFA - IDR - eGFP SPT5 - CTR - mCherry clusters in vitro . arrowhead represents bleached recovered cluster . I Normalized FRAP curves for NELFA - IDR - eGFP SPT5 - CTR - mCherry clusters . bleaching events occurred at 0 second . Results shown are from two biological replicates .

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H 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.


A Western blot 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 .

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A 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 labeled by EU in HEK - 293T expressing SPT5 , SPT5ΔNTR or SPT5ΔCTR after SPT5 depletion .

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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 occupancies of Pol II at promoters gene bodies of GAPDH ( G ) , MYC ( H ) in HEK - 293T expressing SPT5 , SPT5ΔNTR or SPT5ΔCTR after SPT5 depletion . 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 - , unpaired Student ' s t 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 , 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 , p value ( from left to right , 0 . 0004 , 0 . 0013 , 0 . 0081 , 0 . 0299 , 0 . 1332 , 0 . 5839 , 0 . 4404 , 0 . 6170 ) .

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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).


B microscopy showing phase - separated AFF4 - IDR - eGFP SPT5 - CTR - mCherry droplets formed in 50 mM NaCl containing buffer in presence of 10 % PEG - 8000 . ratios are indicated as CAFF4 - IDR / CSPT5 - CTR . C Dot plot showing droplet in Fig 4B . Fields per condition n = 5 .

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B 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. D


D Live confocal showing FRAP of heterotypic eGFP - AFF4 + ENL mCherry - SPT5 droplets . arrowhead represents bleached recovered droplet . E Normalized FRAP curves for eGFP - AFF4 + ENL ( green ) mCherry - SPT5 ( purple ) in AFF4 / ENL / SPT5 heterotypic droplets . bleaching events occurred at 0 second . Results shown are from three biological replicates .

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D 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.


C Confocal imaging of FOS FISH with concurrent SPT5 IF in wild type ( WT ) ENL KO HCT116 after serum treatment . Zoomed - in views of merged regions are indicated by arrow .

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C 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 showing SPT5 in in WT or ENL KO treatment with KL - 1 . I Violin plot showing number of containing in Fig 5H . Results are representative of three biological replicates , each n > 20 . Two - , unpaired Student ' s t was performed . * * * p < 0 . 001 .

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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 showing co - localization of Pol II - S5P with SPT5 in in KL - 1 treated HCT 116 . Results shown are representative from three independent experiments . ( K ) Boxplots showing mean values of Pearson correlation coefficient of co - localization ratios of SPT5 Pol II - S5P in Fig 5J . Central band is median ; boxes represent 1st 3rd quartile ( 25th 75th percentile , respectively ) whiskers 1 . 5 × interquartile range . Results are representative of three biological replicates , each n > 20 . Two - , unpaired Student ' s t was performed . KL - 1 vs . DMSO , * * p = 0 . 0050 . ( L ) Same as Fig 5J . but for SPT5 NELFA . ( M ) Same as Fig 5K . but for SPT5 NELFA . Two - , unpaired Student ' s t was performed . KL - 1 vs . DMSO , * p = 0 . 0100 .

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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.


B microscopy showing that 2 μM SPT5 - CTR - eGFP formed phase - separated droplets in NaCl containing buffer . C Droplet number ( top ) droplet ( bottom ) analyses are shown . Central band is median ; boxes represent 1st 3rd quartile ( 25th 75th percentile , respectively ) whiskers 1 . 5 × interquartile range . Results are representative of two biological replicates , each n > 6 . Two - , unpaired Student ' s t 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 , SPT5 - CTR + P - TEFb ( + vs . - ATP )

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

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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.


A Confocal showing co - localization of AFF4 with SPT5 in in AFF4 wild type ( WT ) or R258W . B Dotplot ( left ) showing AFF4 or SPT5 in AFF4 WT or R258W ( Fig 7A ) . Two - , unpaired Student ' s t was performed . AFF4 R258W vs . WT , * p = 0 . 0254 . Violin plot ( right ) showing Pearson correlation coefficient of co - localization ( Fig 7A ) . Each n > 20 . Results are representative of three biological replicates . Two - , unpaired Student ' s t was performed . AFF4 R258W vs . WT , * p = 0 . 0166 .

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A 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. C


C Droplet experiments showing that CHOPS syndrome missense mutations reinforced AFF4 - ALF domain droplet formation . D Dotplot showing droplet areas in Fig 7C . Fields per condition n = 5 . Two - , unpaired Student ' s t 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 for experiments in Fig 7C . Error bars represent standard deviations . Fields per condition n = 5 . Two - , unpaired Student ' s t was performed . T254S , T254A or R258W vs . WT , * p < 0 . 05 , * * p < 0 . 01 .

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C 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. F


K - N ChIP - qPCR showing occupancies of Pol II ( K ) , AFF4 ( L ) , SPT5 ( M ) , PAF1 ( N ) at promoter gene of HSP70 in AFF4 WT R258W HEK - 293T after heat shock for 2 hours . 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 - , unpaired Student ' s t was performed . For Pol II ChIP , HSP70 _ promoter , * * * p < 0 . 0001 ; HSP70 _ gene , * * * p = 0 . 0002 . For AFF4 ChIP , HSP70 _ promoter , * * * p < 0 . 0001 ; HSP70 _ gene , * * * p < 0 . 0001 . For SPT5 ChIP , HSP70 _ promoter , * * * p < 0 . 0001 ; HSP70 _ gene , * * p = 0 . 0086 . For PAF1 ChIP , HSP70 _ promoter , * * * p = 0 . 0007 ; HSP70 _ gene , * * p = 0 . 0021 .

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K-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.


Western blot quantitation of Sel1L - Hrd1 ERAD in livers of WT mice at 3 , 9 24 of ( n = 3 - 6 per group , 2 independent repeats ) . α - tubulin , loading controls for Western blot .

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Western 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 quantitation of Sel1L - Hrd1 ERAD in 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 .

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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 quantitation of ERAD in Sel1Lf / f Sel1LAlb livers ( n = 4 per group , 3 independent repeats ) . Hsp90 loading controls for Western blot .

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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 ( n = 10 each ) ( n = 7 each ) mice .

Growth curves of male (n=10 each) and female (n=7 each) mice.


Representative ( E ) nose - to - anus ( F ) of mice at 6 of ( n = 6 - 10 per group ) .

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Representative images (E) and nose-to-anus length (F) of male mice at 6 weeks of age (n=6-10 per group).


Organ - to - - ratios of liver kidney in 6 - week - old mice ( n = 6 per group , 3 independent repeats ) .

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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 ( gbw ) ( n = 3 per group , measured over 3 days ) .

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 ( n = 6 per group ) .

Representative estrus cycle mapping in 2-4-month old females (n=6 per group).


H & E of paraffin - embedded liver sections from 9 - week - old mice ( n = 4 per group , 3 independent repeats ) .

H&E images of paraffin-embedded liver sections from 9-week-old mice (n=4 per group, 3 independent repeats).


Western blot of ( 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 .

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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 ( ) obtained from 9 - week - old mice livers ( n = 10 - 12 from one mouse each ) . N , nucleus ; mito , mitochondria ; ER , endoplasmic reticulum .

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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.


Volcano plot depicting transcriptomics data from livers of 9 - week - old Sel1Lf / f Sel1LAlb mice ( n = 3 per group ) ; dotted marks p = 0 . 05 ; black dots represent fold change > 2 .

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Volcano 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 .

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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 .

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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 of Fgf21 in serum from 8 - 9 - week - old mice ( n = 6 - 7 per group ) .

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ELISA analysis of Fgf21 in serum from 8-9-week-old mice (n=6-7 per group).


of Sel1L ( left panel ) mRNA of Fgf21 ( right panel ) in mouse hepatocytes isolated from tamoxifen - inducible Sel1L - knockout Sel1LERCre mice ( 2 independent repeats ) . Hsp90 , loading control for Western blot . Ribosomal L32 , loading control for qPCR .

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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 of hepatic control adipose Sel1L ( n = 3 per group ) ; Hsp90 loading control for Western blot .

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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 of hepatic Fgf21 expression ELISA of serum Fgf21 ( n = 3 per group , 2 independent repeats ) . Ribosomal L32 , loading control for qPCR .

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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 downregulated genes in Fgf21 Tg livers their expression in Sel1LAlb livers ( n = 3 per group ) .

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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 logarithmic fold - change ( FC ) for 16 , 402 genes in Sel1LAlb Fgf21 transgenic ( Tg ) livers ( n = 3 per group ) ; genes that are highly upregulated or downregulated in both datasets are marked in red blue , respectively ; genes that are upregulated unique to each data set ( e . g . Derl3 for Sel1L - Hrd1 ERAD - deficient liver ) are marked in green .

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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 Sel1LAlb mice were injected i . v . with AAV8 - shFgf21 or control AAV8 - shLuc : qPCR of Fgf21 mRNA 3 after injection ( n = 4 per group ) . Ribosomal L32 , loading control for qPCR .

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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 Sel1LAlb mice were injected i . v . with AAV8 - shFgf21 or control AAV8 - shLuc : ELISA of Fgf21 in serum ( K ) 3 after injection ( n = 4 per group ) .

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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 Sel1LAlb mice were injected i . v . with AAV8 - shFgf21 or control AAV8 - shLuc : gain curve after injection ( n = 7 per group ) .

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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).


Western blot of p - Stat5 in livers of 9 - week - old mice ( n = 3 per group , 3 independent repeats ) , with quantitation of of p - to total Stat5 shown below blot . Hsp90 , loading control for Western blot .

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Western 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 of p - Stat5 - associated growth genes in livers of 9 - week - old mice ( n = 6 per group , 2 independent repeats ) . Ribosomal L32 , loading control for qPCR .

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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 mice as measured over 24 hr ( n = 4 per group ) .

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Z ambulatory activity of 9-week-old male mice as measured over 24 hr (n=4 males per group).


Blood glucose in 9 - week - old mice after 6 hr fasting ( n = 6 - 10 per group ) .

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Blood glucose levels in 9-week-old mice after 6 hr fasting (n=6-10 per group).


serum insulin 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 ) in 9 - week - old mice after 6 hr fasting ( n = 6 - 10 per group ) .

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serum triglyceride (TG) levels in 9-week-old mice after 6 hr fasting (n=6-10 per group).


serum cholesterol ( CHOL ) 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 of inguinal adipose tissue ( iWAT ) from 8 - week - old mice ( n = 3 per group , 2 independent repeats ) .

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H&E images of inguinal white adipose tissue (iWAT) from 8-week-old mice (n=3 per group, 2 independent repeats).


qPCR of browning - related genes in iWAT ( n = 3 - 6 per group ) . Ribosomal L32 , loading control for qPCR .

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qPCR analysis of browning-related genes in iWAT (n=3-6 per group). Ribosomal L32, loading control for qPCR analysis.


Western blot of Ucp1 in iWAT of 8 - week - old mice ( n = 3 - 4 per group , 3 independent repeats ) . Hsp90 , loading control for Western blot .

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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.


gain curve of mice after 60 % high fat diet ( HFD ) starting at 5 of ( n = 4 per group , 2 independent repeats ) .

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 normalized to in mice following 9 of HFD feeding ( n = 4 per group ) .

Adipose tissue weight normalized to body weight in male mice following 9 weeks of HFD feeding (n=4 per group).


Western blot of Crebh , Crebh - N , Pparα Xbp1s in whole lysates ( A ) extracts ( B ) in WT Sel1LAlb livers ( n = 3 per group , 3 independent repeats ) with quantitation shown in ( C ) . Hsp90 lamin , loading controls for Western blot .

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Western 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 of Crebh , Ppara Xbp1s in WT Sel1LAlb livers ( n = 4 - 6 per group , 2 independent repeats ) . Ribosomal L32 , loading control for qPCR .

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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 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 .

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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 of Crebh in 9 - week - old mice ( n = 3 per group , 2 independent repeats ) . WT mice injected i . p . with tunicamycin ( Tm , 1 . 5 µg / g ) for 72 hours were included as a control . Hsp90 loading controls for Western blot .

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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 of Crebh in livers of 9 - week - old mice after NP40 - detergent fractionation into NP40 soluble ( NP40S ) pellet ( NP40P ) ( n = 3 per group , 2 independent repeats ) . Hsp90 H2A loading controls for Western blot .

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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 of Crebh in 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 inside in hepatocyte nucleus .

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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 of Crebh binding onto Fgf21 promoter in livers of 9 - week - old mice , normalized first to 5 % input group then to no - antibody ChIP samples ( n = 3 pooled per group , 2 independent repeats ) .

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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 of SEL1L , HRD1 CREBH in human Hep3B hepatocytes upon CRISPR deletion of SEL1L with two different guides . , β - actin loading controls for Western blot .

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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 of SEL1L , HRD1 FGF21 ( K ) in human Hep3B hepatocytes upon CRISPR deletion of SEL1L with two different guides . Ribosomal L32 , loading control for qPCR .

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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.


Western blot of Crebh half - life in transfected WT HRD1 - / - HEK293T treated with cycloheximide ( CHX ) for indicated times . decay of from one experiment is shown below . Hsp90 , loading control for Western blot .

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Western 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 quantitation of Crebh in Crebh - transfected WT ( B ) HRD1 - / - ( C ) HEK293T pre - treated with proteasomal inhibitor bortezomib ( BTZ ) or lysosomal inhibitor chloroquine ( CHQ ) for 2 hours then with CHX for additional 1 hour ( n = 2 per group , 2 independent repeats ) . Hsp90 , loading control for Western blot .

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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 of Crebh ubiquitination following immunoprecipitation ( IP ) of Crebh - Flag Crebh - N - Flag in HEK293T transfected with indicated plasmids . Samples were boiled with SDS before IP for denaturing IP not so for native IP . These were treated with proteasomal inhibitor BTZ for last 6 hr prior to immunoprecipitation . Hsp90 , loading control for Western blot .

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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 of endoglycosidase H ( endoH ) - sensitivity of Crebh in livers of 9 - week - old mice ; ' r ' refers to endoH - cleavage - resistant species ' s ' refers to endoH - cleavage - sensitive species ( n = 3 per group , 2 independent repeats ) . Hsp90 , loading control for Western blot .

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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.


5 - week - old Sel1Lf / f Sel1LAlb mice were injected i . v . once with AAV8 - shCrebh or control AAV8 - shLuc . Western blot of hepatic Sel1L Crebh 5 post injection ( n = 3 mice each , 2 independent repeats ) . Hsp90 , loading control for Western blot .

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5-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.