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 .

O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

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 .

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

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 ) .

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

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 .

O O O O O O B-MEASURED_VAR O O O O B-MEASURED_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 O O O O O O O O

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 .

O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O

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 .

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

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 .

O O O B-MEASURED_VAR O O O O B-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 O O O O O O O O O O O O

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 .

O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 I-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_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

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 )

O O O O O O B-MEASURED_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 O O O O O O O O O O O O O O O O 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 B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR 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 B-CONTROLLED_VAR O O O O O O O

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 .

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

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 .

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

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 .

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

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 .

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

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 .

O O O O B-MEASURED_VAR O B-MEASURED_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

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 .

O O O O B-MEASURED_VAR O B-MEASURED_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 O O O O

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 .

O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

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 ) .

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

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 ) .

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

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 .

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

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 .

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

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 .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-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

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 .

O O O B-MEASURED_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

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 .

O O O O B-MEASURED_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

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 ) .

O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

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 .

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

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 .

O O O O O O O O O O O O O B-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 B-MEASURED_VAR O O O O O O O O O O O O O O O

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 .

O O O O O O O O O O O O O B-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 B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

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 ) .

O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O

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 .

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

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 .

O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR 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 O O O O O O O O O O

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 ) .

O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR 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 O O O O O O

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 ) .

O O O O O O O O O O B-CONTROLLED_VAR O O B-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

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 .

O O O O O B-MEASURED_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 B-MEASURED_VAR O O O O O O O O O O O O

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 .

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

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 ) .

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

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 ) .

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 ) .

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

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 ) .

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

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 .

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 .

O O O B-MEASURED_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

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 .

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

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 .

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

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 .

O O O B-MEASURED_VAR O O B-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

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 .

O O O B-MEASURED_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 O O O O O O O O O O O O O

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 .

O O O B-MEASURED_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 O O O

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 .

O O O B-MEASURED_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 B-MEASURED_VAR O O O O O

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 ) .

O O B-MEASURED_VAR O O B-MEASURED_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

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 .

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

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 .

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

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 .

O O O B-MEASURED_VAR O O O O O O B-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

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 .

O O O O B-MEASURED_VAR O O O O O O O O B-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 O O O O O O O O O O O O O

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 .

O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O B-MEASURED_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 O O O O O O O O O

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 .

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

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 .

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

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.


5 - week - old Sel1Lf / f Sel1LAlb mice were injected i . v . once with AAV8 - shCrebh or control AAV8 - shLuc . qPCR of Crebh Fgf21 mRNA 5 post injection ( n = 3 per group , 2 independent repeats ) . Ribosomal L32 , loading control for qPCR .

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

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 Sel1LAlb mice were injected i . v . once with AAV8 - shCrebh or control AAV8 - shLuc . ELISA of circulating Fgf21 ( C ) 5 post injection ( n = 3 per group , 2 independent repeats ) .

O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR 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 O O O O O O O O

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 Sel1LAlb mice were injected i . v . once with AAV8 - shCrebh or control AAV8 - shLuc . qPCR of hepatic growth - associated genes 5 after injection ( n = 6 per group ) . Ribosomal L32 , loading control for qPCR .

O O O O O B-CONTROLLED_VAR O O B-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 O O O

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 Sel1LAlb mice were injected i . v . once with AAV8 - shCrebh or control AAV8 - shLuc . gain 6 post injection ( n = 10 per group ) .

O O O O O B-CONTROLLED_VAR O O B-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

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 Sel1LAlb mice were injected i . v . once with AAV8 - shCrebh or control AAV8 - shLuc . Insulin tolerance ( ITT ) 5 - after injection ( n = 10 per group ) .

O O O O O B-CONTROLLED_VAR O O B-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

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 Sel1LAlb mice were injected i . v . once with AAV8 - shCrebh or control AAV8 - shLuc . qPCR of Ucp1 in inguinal adipose tissue ( iWAT ) 5 post injection ( n = 3 per group , 2 independent repeats ) . Ribosomal L32 , loading control for qPCR .

O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O

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 Sel1LAlb mice were injected i . v . once with AAV8 - shCrebh or control AAV8 - shLuc . Western blot ( H ) of Ucp1 in inguinal adipose tissue ( iWAT ) 5 post injection ( n = 3 per group , 2 independent repeats ) . Hsp90 , loading control for Western blot .

O O O O O B-CONTROLLED_VAR O O B-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 B-MEASURED_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

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.


of correlation between Sel1L - Hrd1 ERAD Crebh - Fgf21 during fasting - feeding Western blot of hepatic Sel1L - Hrd1 ERAD Crebh of ( Nuc ) ( Cyto ) fractions from 10 - week - old Sel1Lf / f Sel1LAlb mice under overnight fasted or fed states .

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

Analysis 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.


of correlation between Sel1L - Hrd1 ERAD Crebh - Fgf21 during fasting - feeding Quantitation of of Sel1L / Hrd1 / Crebh Fgf21 mRNA in livers of 10 - week - old Sel1Lf / f mice .

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

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 ) in livers of 10 - week - old Sel1Lf / f Sel1LAlb mice .

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

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 in 10 - week - old Sel1Lf / f Sel1LAlb mice .

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

during fasting-feeding Serum Fgf21 levels in 10-week-old Sel1Lf/f and Sel1LAlb mice.


of correlation between Sel1L - Hrd1 ERAD Crebh - Fgf21 during growth Western blot of hepatic Sel1L - Hrd1 ERAD Crebh of ( Nuc ) ( Cyto ) fractions from livers from Sel1Lf / f Sel1LAlb mice at 3 , 9 24 of .

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

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.


of correlation between Sel1L - Hrd1 ERAD Crebh - Fgf21 during growth Quantitation of of Sel1L / Hrd1 / Crebh Fgf21 mRNA with livers from Sel1Lf / f Sel1LAlb mice at 3 , 9 24 of .

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

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 ) in livers from Sel1Lf / f Sel1LAlb mice at 3 , 9 24 of .

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

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.


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

Serum Fgf21 levels in the livers from Sel1Lf/f and Sel1LAlb mice at 3, 9 and 24 weeks of age.


( b ) Upper panel : Representative of BOEC colonies from PBMC cultivation 10 days post - seeding . Dotted in each image outlines a BOEC colony . Lower panel : Stabilized BOEC cultures after passaging of colonies ( scale bar , 250 µm ) . ( c ) Proliferation doubling time for NAFLD healthy BOECs on passages 2 3 . Sample sizes are n = 9 healthy , n = 7 NAFLD ; with 3 biological replicates per donor . Box - whisker plots indicate median ( middle ) , 25th , 75th percentile ( box ) lowest / highest data points ( whiskers ) . ns , not significant ( t - ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Upper panel: Representative images of BOEC colonies from PBMC cultivation 10 days post-seeding. Dotted line in each image outlines a BOEC colony. Lower panel: Stabilized BOEC cultures after passaging of colonies (scale bar, 250 µm). (c) Proliferation doubling time for NAFLD and healthy BOECs based on passages 2 and 3. Sample sizes are n = 9 healthy, n = 7 NAFLD; with 3 biological replicates per donor. Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). ns, not significant (t-test).


( d ) Left panel : Flow cytometry characterization of BOECs for endothelial , leukocyte progenitor markers ( grey - isotype control ; red / blue / green / purple - lineage marker ) . HUVEC monocytic THP - 1 were used as positive controls for endothelial leukocytic markers respectively . Right panel : Percentages of positively expressing . Sample sizes are n = 4 - 6 healthy , n = 2 - 7 NAFLD .

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

(d) Left panel: Flow cytometry characterization of BOECs for endothelial, leukocyte and progenitor cell markers (grey - isotype control; red/blue/green/purple - cell lineage marker staining). HUVEC and monocytic THP-1 cells were used as positive controls for endothelial and leukocytic markers respectively. Right panel: Percentages of positively expressing cells. Sample sizes are n = 4-6 healthy, n = 2-7 NAFLD.


( e ) Representative of immunostaining for endothelial markers , PECAM1 CDH5 ( scale bar , 100 µm ) .

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

(e) Representative images of immunostaining for endothelial markers, PECAM1 and CDH5 (scale bar, 100 µm).


( f ) Left panel : Longitudinal monitoring of BOEC angiogenesis in fibrin gel bead - sprouting assays over 24h ( representative ; scale bar , 100 µm ) . Right panel : Box - whisker plots of quantifications of sprout lengths numbers of sprout per bead at 24h . n = 3 healthy , n = 3 NAFLD , with 4 - 5 beads analyzed per donor 5 - 11 sprouts per bead .

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

(f) Left panel: Longitudinal monitoring of BOEC angiogenesis in fibrin gel bead-based sprouting assays over 24h (representative images; scale bar, 100 µm). Right panel: Box-whisker plots of quantifications of sprout lengths and numbers of sprout per bead at 24h. n = 3 healthy, n = 3 NAFLD, with 4-5 beads analyzed per donor and 5-11 sprouts per bead.


( b ) Volcano plot depicting differentially expressed genes comparing NAFLD BOECs with healthy BOECs . Red blue dots represent upregulated downregulated genes in NAFLD BOECs , respectively .

O O O O O 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) Volcano plot depicting differentially expressed genes comparing NAFLD BOECs with healthy BOECs. Red and blue dots represent upregulated and downregulated genes in NAFLD BOECs, respectively.


( e ) featuring genes in MCODE Cluster 1 .

(e) Heatmap featuring the genes in MCODE Cluster 1.


( a ) Fold change in gene expressions of CXCL10 , CXCL11 , CXCL12 , CCL20 CX3CL1 in NAFLD ( n = 10 ) BOECs , normalized to healthy ( n = 12 ) BOECs , with 2 technical replicates per donor . ( c ) Fold change in gene expressions of CXCL10 , CXCL11 , CXCL12 , CCL20 CX3CL1 in LPS + FFA - treated or non - treated NAFLD ( n = 5 ) healthy ( n = 5 ) BOECs , normalized to non - treated healthy BOECs , with 3 technical replicates per donor .

O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_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 B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_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

(a) Fold change in gene expressions of CXCL10, CXCL11, CXCL12, CCL20 and CX3CL1 in NAFLD (n = 10) BOECs, normalized to healthy (n = 12) BOECs, with 2 technical replicates per donor. (c) Fold change in gene expressions of CXCL10, CXCL11, CXCL12, CCL20 and CX3CL1 in LPS+FFA-treated or non-treated NAFLD (n = 5) and healthy (n = 5) BOECs, normalized to non-treated healthy BOECs, with 3 technical replicates per donor.


( b ) Left : Representative showing non - treated LPS + FFA - treated NAFLD healthy BOECs , stained with Nile Red ( scale bars , 100 µm ) . Right : of intracellular Nile Red stain was quantified at 515 / 585nm , readings were normalized with blanks ( n = 3 donors / group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left: Representative images showing non-treated and LPS+FFA-treated NAFLD and healthy BOECs, stained with Nile Red (scale bars, 100 µm). Right: Fluorescence intensity of intracellular Nile Red stain was quantified at 515/585nm, and readings were normalized with blanks (n = 3 donors/ group).


( d ) concentrations of chemokines in conditioned culture media of healthy NAFLD BOECs . *

(d) Protein concentrations of chemokines in the conditioned culture media of healthy and NAFLD BOECs. *


( e ) Left : Representative ( Bright field ; DAPI , nuclei ; CDH5 , endothelial ; CXCL12 ) of liver sections from chow diet - fed HFHC diet - fed humanized mice ( scale bar , 40 μm ) . Boxed regions were further magnified ( scale bar , 30 µm ) . Right : Box - whisker plot of Mander ' s Coefficient ( M2 ) that was a measure of degree of CXCL12 overlap with CDH5 - positive vasculatures in livers . Box - whisker plots indicate median ( middle ) , 25th , 75th percentile ( box ) lowest / highest data points ( whiskers ) . Chow diet - fed ( n = 4 ) HFHC diet - fed ( n = 4 ) humanized mice were analysed , with 10 - 20 independent regions of interest per mouse were analysed for image .

O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_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 O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(e) Left: Representative images (Bright field; DAPI, nuclei; CDH5, endothelial cells; CXCL12) of liver sections from chow diet-fed and HFHC diet-fed humanized mice (scale bar, 40 μm). Boxed regions were further magnified (scale bar, 30 µm). Right: Box-whisker plot of Mander's Coefficient (M2) that was a measure of the degree of CXCL12 overlap with CDH5-positive vasculatures in the livers. Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). Chow diet-fed (n = 4) and HFHC diet-fed (n = 4) humanized mice were analysed, with 10-20 independent regions of interest per mouse were analysed for image quantification.


( f ) Left : Representative ( Bright field ; DAPI , nuclei ; CDH5 , endothelial ; CXCL12 ) of aortic sections from chow diet - fed LIDPAD mice ( scale bar , 40 μm ) . Boxed regions were further magnified ( scale bar , 30 µm ) . Arrows point at aortic endothelial . AW : Aortic wall ; L : lumen . Right : Box - whisker plot of CXCL12 in aortic vascular endothelia . CXCL12 for each delimitated endothelial region is represented as data points on plot . Box - whisker plots indicate median ( middle ) , 25th , 75th percentile ( box ) lowest / highest data points ( whiskers ) . Chow diet - fed ( n = 3 ) LIDPAD diet - fed ( n = 3 ) mice were analysed , with 10 - 20 delimitated endothelial regions per mouse analysed for image .

O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_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 O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(f) Left: Representative images (Bright field; DAPI, nuclei; CDH5, endothelial cells; CXCL12) of aortic sections from chow diet-fed and LIDPAD mice (scale bar, 40 μm). Boxed regions were further magnified (scale bar, 30 µm). Arrows point at aortic endothelial cells. AW: Aortic wall; L: lumen. Right: Box-whisker plot of CXCL12 fluorescent intensity in the aortic vascular endothelia. CXCL12 fluorescent intensity for each delimitated endothelial region is represented as individual data points on the plot. Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). Chow diet-fed (n = 3) and LIDPAD diet-fed (n = 3) mice were analysed, with 10-20 delimitated endothelial regions per mouse analysed for image quantification.


( g ) Left : Representative showing aortic sections harvested from mice fed with either chow or LIDPAD diet . Sections were stained for leukocyte markers CD45 ( red ) to determine extent of leukocyte infiltration . Dotted lines demarcate aortic walls ( AW ) . L : lumen . Scale bar : 20 µm . Right : Box - whisker plot of mean ( MFI ) of CD45 signals as a readout for amount of infiltrated CD45 + leukocytes within aortic walls . Box - whisker plots indicate median ( middle ) , 25th , 75th percentile ( box ) lowest / highest data points ( whiskers ) . n = 3 mice per group , with 2 tissue sections analyzed per .

O O 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 O O O O O O O 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 B-MEASURED_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 O O O O O

(g) Left: Representative images showing aortic sections harvested from mice fed with either chow or LIDPAD diet. Sections were stained for leukocyte markers CD45 (red) to determine the extent of leukocyte infiltration. Dotted lines demarcate the aortic walls (AW). L: lumen. Scale bar: 20 µm. Right: Box-whisker plot of mean fluorescence intensity (MFI) of CD45 signals as a readout for the amount of infiltrated CD45+ leukocytes within the aortic walls. Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). n = 3 mice per group, with 2 tissue sections analyzed per animal.


( a ) Upper panel : Single - UMAP of sequenced PBMCs from 2 NAFLD 4 healthy subjects ; n = 10 , 786 . We identified 15 distinct types by DICE annotation . Lower panel : Expression patterns of CXCR3 , CXCR4 , ACKR3 , CX3CR1 , CCR6 across different types in NAFLD healthy PBMC samples .

O O O O O O O O O O O O O O O O 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 B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O

(a) Upper panel: Single-cell UMAP of sequenced PBMCs from 2 NAFLD patients and 4 healthy subjects; n = 10,786 cells. We identified 15 distinct cell types by DICE annotation. Lower panel: Expression patterns of CXCR3, CXCR4, ACKR3, CX3CR1, CCR6 across different cell types in NAFLD and healthy PBMC samples.


( c ) Graphic shows composition of identified types in NAFLD healthy PBMCs single - .

(c) Graphic shows the percentage composition of identified cell types in NAFLD and healthy PBMCs single-cell analysis.


( d ) Upper panel : Representative flow cytometry dot plots , characterizing isolated CD8 + T CD8 - immune fraction ( mainly CD4 + T , NK monocytes ) from PBMCs of healthy NAFLD subjects . Lower panel : Flow cytometry of % of positively expressing CXCR4 + CX3CR1 + within each immune subset . Sample sizes are n = 14 NAFLD ; n = 6 healthy .

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 O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

(d) Upper panel: Representative flow cytometry dot plots, characterizing the isolated CD8+ T cells and CD8- immune cell fraction (mainly CD4+ T cells, NK cells and monocytes) from PBMCs of healthy and NAFLD subjects. Lower panel: Flow cytometry quantification of % of positively expressing CXCR4+ and CX3CR1+ cells within each immune subset. Sample sizes are n = 14 NAFLD; n = 6 healthy.


( e ) of gene expressions of endothelial antigens relevant to interaction with T lymphocytes in NAFLD ( n = 3 ) healthy ( n = 3 ) BOECs , with 3 biological replicates per donor .

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

(e) Heatmap of gene expressions of endothelial antigens relevant to interaction with T lymphocytes in NAFLD (n = 3) and healthy (n = 3) BOECs, with 3 biological replicates per donor.


( f ) Upper panel : Representative flow cytometry histograms of HLA - A / B / C , HLA - DM IFN - γR1 expressions in NAFLD ( red ) healthy ( blue ) BOECs . Grey , isotype control . Lower panel : Box - whisker plots reflect mean ( a . u . , arbituary unit ) , normalized to isotype control , as a readout for epitope density . Box - whisker plots indicate median ( middle ) , 25th , 75th percentile ( box ) lowest / highest data points ( whiskers ) . ns , non - significant ( t - ) . n = 4 donors ; * p < 0 . 05 ; ns , not significant ( Mann - Whitney for HLA - A / B / C IFN - γR1 ; t - for HLA - DM ) .

O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O

(f) Upper panel: Representative flow cytometry histograms of HLA-A/B/C, HLA-DM and IFN-γR1 expressions in NAFLD (red) and healthy (blue) BOECs. Grey, isotype control. Lower panel: Box-whisker plots reflect mean fluorescence intensity (a.u., arbituary unit), normalized to isotype control, as a readout for epitope density. Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). ns, non-significant (t-test). n = 4 donors; *p<0.05; ns, not significant (Mann-Whitney test for HLA-A/B/C and IFN-γR1; t-test for HLA-DM).


( a ) Left : Workflow schematic of adhesion . Middle : Representative of labeled THP - 1 Jurkat attached to BOEC monolayers ( Scale bars , 100 µm ) . Right : Box - whisker plots of numbers of adhered leukocytes were determined fluorometrically . Sample sizes are n = 9 NAFLD n = 7 healthy , with 3 biological replicates per donor .

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

(a) Left: Workflow schematic of adhesion assay. Middle: Representative images of labeled THP-1 and Jurkat cells attached to BOEC monolayers (Scale bars, 100 µm). Right: Box-whisker plots of the numbers of adhered leukocytes were determined fluorometrically. Sample sizes are n = 9 NAFLD and n = 7 healthy, with 3 biological replicates per donor.


( b ) Left : Schematic of chemotaxis . Right : Box - whisker plots of numbers of recruited THP - 1 Jurkat by BOECs . Box - whisker plots indicate median ( middle ) , 25th , 75th percentile ( box ) lowest / highest data points ( whiskers ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left: Schematic of chemotaxis assay. Right: Box-whisker plots of the numbers of recruited THP-1 and Jurkat cells by the BOECs. Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers).


( c ) CD8 + T CD8 - fractions were isolated from NAFLD healthy subjects . Box - whisker plots show number of recruited immune to their respective BOECs matched by health condition in coculture chemotaxis assays . Box - whisker plots indicate median ( middle ) , 25th , 75th percentile ( box ) lowest / highest data points ( whiskers ) . ns , non - significant ( t - ) . Sample sizes are n = 11 NAFLD n = 6 healthy , with 3 - 4 technical replicates per donor .

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

(c) CD8+ T cells and CD8- cell fractions were isolated from NAFLD and healthy subjects. Box-whisker plots show the number of recruited immune cells to their respective BOECs matched by health condition in coculture chemotaxis assays. Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). ns, non-significant (t-test). Sample sizes are n = 11 NAFLD and n = 6 healthy, with 3-4 technical replicates per donor.


( d ) Box - whisker plots show percentages of inhibition on immune chemotaxis by treatment with AMD3100 ( CXCR4 small molecule inhibitor ) . Box - whisker plots indicate median ( middle ) , 25th , 75th percentile ( box ) lowest / highest data points ( whiskers ) . ns , non - significant ( t - ) . Sample sizes are n = 6 NAFLD n = 4 healthy , with 3 - 4 technical replicates per donor .

O O O O O O O O O O O O O O O O O O O B-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 O O O O O O O O O O O O O O O O O O O O O

(d) Box-whisker plots show percentages of inhibition on immune cells chemotaxis by treatment with AMD3100 (CXCR4 small molecule inhibitor). Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). ns, non-significant (t-test). Sample sizes are n = 6 NAFLD and n = 4 healthy, with 3-4 technical replicates per donor.


( e ) Box - whisker plots show percentages of inhibition on immune chemotaxis by treatment with anti - CXCL12 IgG ( CXCL12 neutralizing antibody ) . Box - whisker plots indicate median ( middle ) , 25th , 75th percentile ( box ) lowest / highest data points ( whiskers ) . ns , non - significant ( t - ) . Sample sizes are n = 5 NAFLD n = 3 healthy , with 3 - 4 technical replicates per donor .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-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 O O O O O O O O O O O O O O O O O O O O

(e) Box-whisker plots show percentages of inhibition on immune cells chemotaxis by treatment with anti-CXCL12 IgG (CXCL12 neutralizing antibody). Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). ns, non-significant (t-test). Sample sizes are n = 5 NAFLD and n = 3 healthy, with 3-4 technical replicates per donor.


( f ) Left : Schematic of transendothelial migration with - derived BOECs immune . Right : Transendothelial electric resistance ( TEER ) measurements of BOEC barrier tightness in presence absence of PBMCs / or AMD3100 . Box - whisker plots indicate median ( middle ) , 25th , 75th percentile ( box ) lowest / highest data points ( whiskers ) . ns , non - significant ( t - ) . Sample is n = 5 samples , with 3 technical TEER readings recorded per sample .

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

(f) Left: Schematic of transendothelial migration assay with patient-derived BOECs and immune cells. Right: Transendothelial electric resistance (TEER) measurements of BOEC barrier tightness in the presence and absence of PBMCs and/or AMD3100. Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). ns, non-significant (t-test). Sample size is n = 5 patient samples, with 3 technical TEER readings recorded per sample.


( g ) Box - whisker plots of numbers of circulating endothelial per million PBMCs in healthy ( n = 6 ) NAFLD subjects ( n = 11 ) . Box - whisker plots indicate median ( middle ) , 25th , 75th percentile ( box ) lowest / highest data points ( whiskers ) . ns , non - significant ( t - ) .

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

(g) Box-whisker plots of the numbers of circulating endothelial cells per million PBMCs in healthy (n = 6) and NAFLD subjects (n = 11). Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). ns, non-significant (t-test).


( A ) . A FRET was used to measure difference in melting ( ΔTm ) of ( G4C2 ) 4 or G - Qs in presence of 2 μM of 138 different small molecules . An increase in ΔTm indicates stabilisation of G - Q . Small molecules are ranked on x axis according to their increasing thermal stabilisation of ( G4C2 ) 4 G - Q . Small molecules that preferentially stabilise over ( G4C2 ) 4 G - Qs reside in upper part of scatter plot , above blue curve . An arbitrary ΔTm threshold of 13 ° C greater than vehicle ( grey ) a differential binding to over ( ΔTmRNA - ΔTmDNA ≥ 5 ° C ) were used to select candidate small molecules .

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

(A). A FRET assay was used to measure the difference in melting temperature (ΔTm) of (G4C2)4 RNA or DNA G-Qs in the presence of 2 μM of 138 different small molecules. An increase in ΔTm indicates stabilisation of the G-Q. Small molecules are ranked on the x axis according to their increasing thermal stabilisation of the DNA (G4C2)4 G-Q. Small molecules that preferentially stabilise RNA over DNA (G4C2)4 G-Qs reside in the upper part of the scatter plot, above the blue curve. An arbitrary ΔTm threshold of 13 °C greater than vehicle (grey line) and a differential binding to RNA over DNA (ΔTmRNA-ΔTmDNA ≥ 5 °C) were used to select candidate small molecules.


( C ) . FRET dose - response of DB1246 , DB1247 DB1273 on stabilisation of or ( G4C2 ) 4 G - Qs .

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

(C). FRET dose-response of DB1246, DB1247 and DB1273 on stabilisation of RNA or DNA (G4C2)4 G-Qs.


( D ) . unfold CD spectra for ( G4C2 ) 4 alone ( which shows a characteristic G - Q structure with minima at 237 nm , maxima at 264 nm no additional signal ) , or in presence of 2 μM DB1246 , DB1247 or DB1273 . A characteristic induced CD spectrum , in 350 nm - 550 nm region , is observed only in presence of each small molecule , confirming that each of these three compounds are binding to ( G4C2 ) 4 G - Qs . Data in A represent mean ± s . d . , n = 1 with 3 technical replica . Data in C represent mean ± s . d . , n = 3 independent experiments . Data in D represent mean ± s . d . , n = 3 independent experiments .

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

(D). Temperature unfold CD spectra for (G4C2)4 RNA alone (which shows a characteristic G-Q structure with minima at 237 nm, maxima at 264 nm and no additional signal), or in the presence of 2 μM DB1246, DB1247 or DB1273. A characteristic induced CD spectrum, in the 350 nm - 550 nm region, is observed only in the presence of each small molecule, confirming that each of these three compounds are binding to (G4C2)4 RNA G-Qs. Data in A represent mean ± s.d., n=1 with 3 technical replica. Data in C represent mean ± s.d., n=3 independent experiments. Data in D represent mean ± s.d., n=3 independent experiments.


( A ) Representative of C9orf72 iPSC - cortical neurons treated with DMSO ( vehicle control ) or 1 µM of DB1246 , DB1247 or DB1273 , for 4 days . foci are shown in red nuclei ( DAPI ) in blue . Scale bar represents 10 µm .

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

(A) Representative images of C9orf72 iPSC-cortical neurons treated with DMSO (vehicle control) or 1 µM of DB1246, DB1247 or DB1273, for 4 days. RNA foci are shown in red and nuclei (DAPI) in blue. Scale bar represents 10 µm.


( B ) shows foci are significantly reduced by three compounds in iPSC - cortical neurons by DB1246 DB1273 in iPSC - motor neurons . Data are shown as average of neurons containing foci relative to vehicle ( DMSO ) . N = 3 independent C9orf72 lines with 2 - 3 inductions per at least 70 neurons counted per induction . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , one sample two - t - versus normalised control . For cortical neurons , p = 0 . 0124 ( DB1246 ) , p = 0 . 0065 ( DB1247 ) , p = 0 . 0096 ( DB1273 ) . For motor neurons , p = 0 . 0004 ( DB1246 ) , p = 0 . 0030 ( DB1273 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification shows RNA foci are significantly reduced by all three compounds in iPSC-cortical neurons and by DB1246 and DB1273 in iPSC-motor neurons. Data are shown as the average percentage of neurons containing RNA foci relative to vehicle (DMSO). N=3 independent C9orf72 patient lines with 2-3 inductions per line and at least 70 neurons counted per induction. *p <0.05, **p <0.01, ***p <0.001, one sample two-tailed t-test versus normalised control. For cortical neurons, p=0.0124 (DB1246), p=0.0065 (DB1247), p=0.0096 (DB1273). For motor neurons, p=0.0004 (DB1246), p=0.0030 (DB1273).


( A ) Poly ( GP ) were measured by MSD immunoassay in C9orf72 iPSC - motor neurons treated for 7 days with 8 - 16 µM of G4C2 repeat G - Q binding small molecules DB1247 , DB1247 DB1273 . Treatment with DB1246 or DB1273 leads to a significant reduction in poly ( GP ) relative to vehicle - treated controls . Data are shown as mean of 3 independent C9orf72 iPSC lines , with 1 - 6 differentiations per . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , one sample two - t - versus normalised control . p = 0 . 0068 ( DB1246 , 12 µm ) , p = 0 . 0417 ( DB1246 , 16 µm ) , p = 0 . 0196 ( DB1247 , 8 µm ) , p = 0 . 0002 ( DB1273 , 8 µm ) , p = 0 . 0194 ( DB1273 , 12 µm ) , p = 0 . 0062 ( DB1273 , 16 µm )

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 O O O O O O O O O O O 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 I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Poly(GP) levels were measured by MSD immunoassay in C9orf72 patient iPSC-motor neurons treated for 7 days with 8-16 µM of the G4C2 repeat G-Q binding small molecules DB1247, DB1247 and DB1273. Treatment with DB1246 or DB1273 leads to a significant reduction in poly(GP) levels relative to vehicle-treated controls. Data are shown as the mean and SEM of 3 independent C9orf72 iPSC lines, with 1-6 differentiations per line. *p<0.05, **p<0.01, *** p<0.001, one sample two-tailed t-test versus normalised control. p=0.0068 (DB1246, 12 µm), p=0.0417 (DB1246, 16 µm), p=0.0196 (DB1247, 8 µm), p=0.0002 (DB1273, 8 µm), p=0.0194 (DB1273, 12 µm), p=0.0062 (DB1273, 16 µm)


( B ) C9orf72 iPSC - motor neurons were treated for 7 days with 16 µM of each small molecule expression of MCM2 three C9orf72 transcript variants ( V1 - V3 ) measured by quantitative RT - PCR . Data are shown as mean s . d . of 3 independent iPSC - motor neuron lines ( one induction per ) relative to vehicle ( DMSO ) treated controls . No significant changes in gene expression were observed , one way ANOVA , Dunnett ' s post hoc , p > 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_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 O O O O O 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) C9orf72 patient iPSC-motor neurons were treated for 7 days with 16 µM of each small molecule and the expression levels of MCM2 and the three C9orf72 transcript variants (V1-V3) measured by quantitative RT-PCR. Data are shown as the mean and s.d. of 3 independent iPSC-motor neuron lines (one induction per line) relative to vehicle (DMSO) treated controls. No significant changes in gene expression were observed, one way ANOVA, Dunnett's post hoc test, p>0.05.


( C ) XTT for C9orf72 iPSC - motor neurons treated for 7 days with 0 - 40 µM of DB1246 , DB1247 , DB1273 or cisplatin as a positive control . Data are shown as mean of two independent iPSC - motor neuron lines . Toxicity is only observed at highest dose of 40 µM not at penultimate dose of 20 µM .

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

(C) XTT cell death assay for C9orf72 iPSC-motor neurons treated for 7 days with 0 - 40 µM of DB1246, DB1247, DB1273 or cisplatin as a positive control. Data are shown as the mean and SEM of two independent iPSC-motor neuron lines. Toxicity is only observed at the highest dose of 40 µM and not at the penultimate dose of 20 µM.


( D ) Drosophila first instar larvae expressing 36 G4C2 repeats were placed on food containing either vehicle or 1 mM DB1273 , number reaching pupal stage of development counted after 7 days . DB1273 treatment ( n = 1200 flies ) significantly improves survival compared to vehicle ( n = 1150 flies ) , p = 0 . 0320 , chi - squared . Data are shown as proportion reaching pupal stage , with numbers within each group indicated on bars . Genotype was w1118 ; ( GGGGCC ) 36 / + ; daGAL4 / + ( daGAL4 > 36R ) .

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

(D) Drosophila first instar larvae expressing 36 G4C2 repeats were placed on food containing either vehicle or 1 mM DB1273, and the number reaching the pupal stage of development counted after 7 days. DB1273 treatment (n=1200 flies) significantly improves survival compared to vehicle (n=1150 flies), p=0.0320, chi-squared test. Data are shown as proportion reaching the pupal stage, with numbers within each group indicated on the bars. Genotype was w1118; (GGGGCC)36/+; daGAL4/+ (daGAL4>36R).


( E ) Drosophila first instar larvae ubiquitously expressing 36 G4C2 repeats ( 36R ) were treated with vehicle or 1 mM DB1273 for 5 days , until third instar stage ( L3 ) , poly ( GR ) measured by MSD immunoassay . Poly ( GR ) was also measured in control ( w1118 ) larvae that do not express G4C2 repeats . DB1273 treatment significantly reduced poly ( GR ) expression . n = 8 for 36R groups , n = 7 for control group . * * p < 0 . 01 , * * * p < 0 . 001 , one way ANOVA with Dunnett ' s post hoc . p = 0 . 0001 ( w1118 versus 36R 0 mM larvae ) , p = 0 . 0019 ( 0 mM versus 1 mM 36R larvae ) . Genotypes were w1118 ; ; w1118 ; ( GGGGCC ) 36 / + ; daGAL4 / + ( daGAL4 > 36R ) .

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

(E) Drosophila first instar larvae ubiquitously expressing 36 G4C2 repeats (36R) were treated with vehicle or 1 mM DB1273 for 5 days, until the third instar stage (L3), and poly(GR) measured by MSD immunoassay. Poly(GR) was also measured in control (w1118) larvae that do not express G4C2 repeats. DB1273 treatment significantly reduced poly(GR) expression. n=8 for 36R groups, n=7 for control group. **p<0.01, *** p<0.001, one way ANOVA with Dunnett's post hoc test. p=0.0001 (w1118 versus 36R 0 mM larvae), p=0.0019 (0 mM versus 1 mM 36R larvae). Genotypes were w1118; ; and w1118; (GGGGCC)36/+; daGAL4/+ (daGAL4>36R).


( A ) Coomassie ‐ stained gels showing results of liposome co ‐ sedimentation assays using Folch liposomes indicated . Liposome binding allows to be pelleted . star indicates a degradation product of Atg5 - Atg12 conjugate .

O O O O O O O O O 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 B-MEASURED_VAR O O

(A) Coomassie‐stained gels showing the results of liposome co‐sedimentation assays using Folch liposomes and the indicated proteins. Liposome binding allows the protein to be pelleted. The star indicates a degradation product of the Atg5-Atg12 conjugate.


( B , D ) Yeast of indicated genotype were transformed with plasmids coding for Atg5 Atg16 , respectively , subjected to fractionation experiments . were detected by anti ‐ Myc western blotting . presence of in pellet fraction indicated binding .

O O O O O O O O O O O O O O O B-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

(B, D) Yeast cells of the indicated genotype were transformed with plasmids coding for Atg5 and Atg16, respectively, and subjected to fractionation experiments. The proteins were detected by anti‐Myc western blotting. The presence of the protein in the pellet fraction indicated membrane binding.


( C ) showing relative amounts of Atg5 - 9 × Myc Atg5 - 9 × Myc - Atg12 in pellet fractions relative to Pex30 . amount of Atg5 - 9 × Myc Atg5 - 9 × Myc - Atg12 in atg5Δ was set to 1 . See Supplementary Figure 1 for graph on non ‐ normed data . is on three independent experiments averages standard deviations are shown . numbers next to blots indicate molecular in kDa . P , pellet ; S , supernatant . Figure source data can be found with Supplementary data .

O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O B-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 O O O O O O O O O O O O O O O O

(C) Quantification showing the relative amounts of Atg5-9 × Myc and Atg5-9 × Myc-Atg12 in the pellet fractions relative to Pex30. The amount of Atg5-9 × Myc and Atg5-9 × Myc-Atg12 in atg5Δ cells was set to 1. See Supplementary Figure 1 for graph based on non‐normed data. The quantification is based on three independent experiments and the averages and the standard deviations are shown. The numbers next to the blots indicate the molecular weight in kDa. P, pellet; S, supernatant. Figure source data can be found with the Supplementary data.


( A ) Coomassie ‐ stained gels of liposome co ‐ sedimentation assays using either Atg5 - Atg12 / Atg16 ( upper panel ) or Atg5 / Atg16 ( lower panel ) liposomes with indicated lipid composition ( Folch : Folch lipids , PE / PI3P : 40 % POPC , 35 % POPS , 20 % POPE , 5 % PI3P , PE / PI : 40 % POPC , 35 % POPS , 20 % POPE , 5 % PI , DAG / PI3P : 40 % POPC , 35 % POPS , 20 % DAG , 5 % PI3P , DAG / PI : 40 % POPC , 35 % POPS , 20 % DAG , 5 % PI ) . Note that Atg5 - Atg12 / Atg16 Atg5 / Atg16 complexes show almost identical binding behaviours .

O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O B-MEASURED_VAR O B-MEASURED_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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O

(A) Coomassie‐stained gels of liposome co‐sedimentation assays using either Atg5-Atg12/Atg16 (upper panel) or Atg5/Atg16 (lower panel) and liposomes with the indicated lipid composition (Folch: Folch lipids, PE/PI3P: 40% POPC, 35% POPS, 20% POPE, 5% PI3P, PE/PI: 40% POPC, 35% POPS, 20% POPE, 5% PI, DAG/PI3P: 40% POPC, 35% POPS, 20% DAG, 5% PI3P, DAG/PI: 40% POPC, 35% POPS, 20% DAG, 5% PI). Note that the Atg5-Atg12/Atg16 and the Atg5/Atg16 complexes show almost identical binding behaviours.


( B ) showing amounts of Atg5 - Atg12 / Atg16 Atg5 / Atg16 in pellet fractions . amount of in pellet fraction in absence of liposomes ( background ) was subtracted from amount of in pellet fractions in presence of liposomes . Data were obtained from three independent experiments , one of which is shown in ( A ) . Atg5 - Atg12 Atg5 bands were used for .

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_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 O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O

(B) Quantification showing the amounts of Atg5-Atg12/Atg16 and Atg5/Atg16 in the pellet fractions. The amount of protein in the pellet fraction in the absence of liposomes (background) was subtracted from the amount of protein in the pellet fractions in the presence of liposomes. Data were obtained from three independent experiments, one of which is shown in (A). The Atg5-Atg12 and Atg5 bands were used for quantification.


( C ) Coomassie ‐ stained gel of a liposome co ‐ sedimentation showing lipid requirements of Atg5 - Atg12 / Atg16 . Liposomes were composed of 40 % POPC , 35 % POPS , 20 % POPE 5 % PI3P . In liposomes containing no POPE or POPS , these lipids were replaced with POPC .

O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_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

(C) Coomassie‐stained gel of a liposome co‐sedimentation assay showing the lipid requirements of the Atg5-Atg12/Atg16 complex. Liposomes were composed of 40% POPC, 35% POPS, 20% POPE and 5% PI3P. In the liposomes containing no POPE or POPS, these lipids were replaced with POPC.


( D ) of Atg5 - Atg12 / Atg16 in pellet fraction in liposome co ‐ sedimentation , one of which is shown in ( C ) . Data were obtained from six independent experiments . Atg5 - Atg12 was used for . average value is shown error bars represent standard deviation . P ‐ values were calculated using Student ' s t ‐ . numbers next to gels indicate molecular in kDa . DAG , diacylglycerol ; P , pellet ; PI , phosphatidylinositol ; PI3P , phosphatidylinositol ‐ 3 ‐ phosphate ; ( PO ) PC , ( 1 ‐ palmitoyl ‐ 2 ‐ oleoyl ‐ ) phosphatidylcholine ; ( PO ) PE , ( 1 ‐ palmitoyl ‐ 2 ‐ oleoyl ‐ ) phosphatidylethanolamine ; ( PO ) PS , ( 1 ‐ palmitoyl ‐ 2 ‐ oleoyl ‐ ) phosphatidylserine ; S , supernatant . Figure source data can be found with Supplementary data .

O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_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 B-MEASURED_VAR O B-MEASURED_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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Quantification of Atg5-Atg12/Atg16 in the pellet fraction in liposome co‐sedimentation, one of which is shown in (C). Data were obtained from six independent experiments. The Atg5-Atg12 was used for quantification. The average value is shown and the error bars represent the standard deviation. P‐values were calculated using Student's t‐test. The numbers next to the gels indicate the molecular weight in kDa. DAG, diacylglycerol; P, pellet; PI, phosphatidylinositol; PI3P, phosphatidylinositol‐3‐phosphate; (PO)PC, (1‐palmitoyl‐2‐oleoyl‐) phosphatidylcholine; (PO)PE, (1‐palmitoyl‐2‐oleoyl‐) phosphatidylethanolamine; (PO)PS, (1‐palmitoyl‐2‐oleoyl‐) phosphatidylserine; S, supernatant. Figure source data can be found with the Supplementary data.


( A ) Coomassie ‐ stained gels showing co ‐ sedimentation assays using Folch liposomes indicated . Atg5 - Atg12 / Atg16 was most efficient in recruiting Atg3 to pellet fraction .

O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O

(A) Coomassie‐stained gels showing co‐sedimentation assays using Folch liposomes and the indicated proteins. Atg5-Atg12/Atg16 was most efficient in recruiting Atg3 to the pellet fraction.


( B ) of Atg3 in pellet fraction in presence of indicated . is on three independent experiments .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O

(B) Quantification of Atg3 in the pellet fraction in the presence of the indicated proteins. The quantification is based on three independent experiments.


( C ) Coomassie ‐ stained gels showing results of Atg8 - PE conjugation assays using indicated E . coli lipids derived liposomes . Atg8 - PE conjugation was detected as characteristic band shift as conjugated form runs faster on urea ‐ SDS gels . numbers above gels indicate time in minutes . Note that at least under these conditions almost no Atg8 - PE conjugation was detected in absence of Atg5 - Atg12 conjugate or Atg5 - Atg12 / Atg16 . numbers next to gels indicate molecular in kDa . averages error bars representing standard deviations are shown . P ‐ values were calculated using Student ' s t ‐ . NS , non ‐ significant ; P , pellet ; S , supernatant . Figure source data can be found with Supplementary data .

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 O B-MEASURED_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 B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_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 O O O O O O O O O O O O O O

(C) Coomassie‐stained gels showing the results of Atg8-PE conjugation assays using the indicated proteins and E. coli lipids derived liposomes. Atg8-PE conjugation was detected as characteristic band shift as the conjugated form runs faster on urea‐SDS gels. The numbers above the gels indicate the time in minutes. Note that at least under these conditions almost no Atg8-PE conjugation was detected in the absence of the Atg5-Atg12 conjugate or the Atg5-Atg12/Atg16 complex. The numbers next to the gels indicate the molecular weight in kDa. The averages and the error bars representing the standard deviations are shown. P‐values were calculated using Student's t‐test. NS, non‐significant; P, pellet; S, supernatant. Figure source data can be found with the Supplementary data.


( A ) Confocal microscopy showing eGFP - Atg8 recruitment to GUVs is dependent on Atg5 - Atg12 / Atg16 PE . was labelled by incorporation of rhodamine ‐ PE .

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

(A) Confocal microscopy images showing eGFP-Atg8 recruitment to GUVs is dependent on the Atg5-Atg12/Atg16 complex and PE. The membrane was labelled by incorporation of rhodamine‐PE.


( B ) Single confocal section showing colocalization of Atg5 - Atg12 / Atg16 - eGFP mCherry - Atg8 on GUVs .

O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O

(B) Single confocal section showing colocalization of Atg5-Atg12/Atg16-eGFP and mCherry-Atg8 on GUVs.


( C ) of GUV ‐ associated eGFP - Atg8 in presence of Atg5 - Atg12 / Atg16 Atg5 - Atg12 conjugate . first graph shows of GUVs associated with eGFP - Atg8 . second graph shows of eGFP - Atg8 on GUVs . is on four independent experiments . Inset : anti ‐ Atg12 western blot using rabbit anti ‐ Atg12 antiserum showing amounts of Atg5 - Atg12 / Atg16 Atg5 - Atg12 conjugate in one of assays used for shown in ( C ) .

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

(C) Quantification of GUV‐associated eGFP-Atg8 in the presence of the Atg5-Atg12/Atg16 complex and the Atg5-Atg12 conjugate. The first graph shows the percentage of GUVs associated with eGFP-Atg8 fluorescence. The second graph shows the intensity of eGFP-Atg8 on the GUVs. The quantification is based on four independent experiments. Inset: anti‐Atg12 western blot using the rabbit anti‐Atg12 antiserum showing the amounts of Atg5-Atg12/Atg16 complex and Atg5-Atg12 conjugate in one of the assays used for the quantification shown in (C).


( D ) of recruitment of Atg3 - mCherry by Atg5 - Atg12 / Atg16 , Atg5 - Atg12 conjugate a buffer control . graph is on two independent experiments .

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

(D) Quantification of membrane recruitment of Atg3-mCherry by the Atg5-Atg12/Atg16 complex, the Atg5-Atg12 conjugate and a buffer control. The graph is based on two independent experiments.


( E ) Widefield showing frames of a time series of GUVs incubated with Atg3 , Atg7 , Atg8 Atg5 - Atg12 / Atg16 ( see Materials methods for details ) . was labelled by incorporation of rhodamine ‐ PE . time is indicated in minutes .

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

(E) Widefield images showing individual frames of a time series of GUVs incubated with Atg3, Atg7, Atg8 and the Atg5-Atg12/Atg16 complex (see Materials and methods for details). The membrane was labelled by incorporation of rhodamine‐PE. The time is indicated in minutes.


( F ) Widefield of GUVs incubated with Atg3 , Atg7 , eGFP - Atg8 Atg5 - Atg12 / Atg16 for 35 min ( left ) or 70 min ( right ) . was labelled by incorporation of rhodamine ‐ PE . Note massive accumulation of eGFP - Atg8 at interface between GUVs . arrows point to some of these interfaces . For quantifications , averages standard deviations are shown . P ‐ values were calculated using Student ' s t ‐ . Scale bars : 10 μm . Figure source data can be found with Supplementary data .

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

(F) Widefield images of GUVs incubated with Atg3, Atg7, eGFP-Atg8 and the Atg5-Atg12/Atg16 complex for 35 min (left) or 70 min (right). The membrane was labelled by incorporation of rhodamine‐PE. Note the massive accumulation of eGFP-Atg8 at the interface between individual GUVs. The arrows point to some of these interfaces. For the quantifications, the averages and standard deviations are shown. P‐values were calculated using Student's t‐test. Scale bars: 10 μm. Figure source data can be found with the Supplementary data.


( A ) Frames of widefield taken from a time series of rhodamine ‐ PE labelled GUVs in presence of Atg5 - Atg12 / Atg16 . For control condition buffer but no was added . Note increase in clustered GUVs over time . numbers indicate time in minutes .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-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

(A) Frames of widefield images taken from a time series of rhodamine‐PE labelled GUVs in the presence of the Atg5-Atg12/Atg16 complex. For the control condition buffer but no protein was added. Note the increase in clustered GUVs over time. The numbers indicate the time in minutes.


( B ) Confocal of rhodamine ‐ PE labelled GUVs incubated with Atg5 - Atg12 / Atg16 - eGFP . Note accumulation of at interface between GUVs ( arrows ) .

O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O

(B) Confocal images of rhodamine‐PE labelled GUVs incubated with Atg5-Atg12/Atg16-eGFP complex. Note the accumulation of the complex at the interface between GUVs (arrows).


( C ) confocal microscopy frames of a time series of rhodamine ‐ PE labelled GUVs incubated with Atg5 - Atg12 / Atg16 - eGFP . arrows point to GUV - GUV interface where initially accumulated .

O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O

(C) Individual confocal microscopy frames of a time series of rhodamine‐PE labelled GUVs incubated with Atg5-Atg12/Atg16-eGFP. The arrows point to the GUV-GUV interface where the protein initially accumulated.


( D ) Confocal microscopy of rhodamine ‐ PE labelled GUVs incubated with Atg5 - Atg12 / Atg16 - eGFP . arrows point to concentrations of Atg5 - Atg12 / Atg16 on GUV . Scale bars : 10 μm ( A - C ) 3 μm ( D ) .

O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

(D) Confocal microscopy images of rhodamine‐PE labelled GUVs incubated with Atg5-Atg12/Atg16-eGFP. The arrows point to the concentrations of Atg5-Atg12/Atg16 on the GUV membrane. Scale bars: 10 μm (A-C) and 3 μm (D).


( A ) Widefield of rhodamine ‐ PE labelled GUVs 30 min GUVs cluster only in presence of Atg5 - Atg12 / Atg16 Atg5 / Atg16 complexes .

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

(A) Widefield images of rhodamine‐PE labelled GUVs 30 min GUVs cluster only in the presence of the Atg5-Atg12/Atg16 and Atg5/Atg16 complexes.