EMBO/SourceData huggingface dataset:
Figure 5Scatter plot with linear regression line and heatmap show log2FC based alterations of the significantly different species in the A) gut microbiome between CMA and placebo groups on Day 70 vs Day 0; B) gut microbiome between Day 70 vs Day 0 in the CMA and placebo groups; C) oral microbiome between CMA and placebo groups on Day 70 vs Day 0; D) oral microbiome between Day 70 vs Day 0 in the CMA and placebo groups. Each dot represents a species and it has been colored according to its corresponding phylum. Asterisks indicate statistical significance based on paired Wilcoxon signed-rank test. p< 0.05. Log2FC: log2(fold change).
Figure 6Heatmap shows the association between the plasma level of clinical variables including liver fat, ALT, AST, uric acid, and creatinine with , B) plasma level of 10 most significant inflammation related proteinsHeatmap shows the association between the plasma level of clinical variables including liver fat, ALT, AST, uric acid, and creatinine with , C) the abundance of the species in gut microbiomeHeatmap shows the association between the plasma level of clinical variables including liver fat, ALT, AST, uric acid, and creatinine with D) the abundance of the species in oral microbiome. AST, Aspartate aminotransferase; ALT, Alanine transaminase. Asterisks indicate statistical significance based on Spearman correlation analysis. p < 0.05; Cor.Coeff: Correlation coefficient.
Figure 1A, B (A) Confocal imaging of FOS DNA FISH with concurrent SPT5 and Pol II-S5P IF showing that SPT5 and Pol II-S5P occupied the FOS loci after serum starvation. Zoomed-in views of the merged regions are indicated by the white arrow. (B) Same as Fig 1A. but for FOS DNA FISH with concurrent SPT5 and NELFE IF.E Confocal images showing that SPT5, NELFA, Pol II-S5P and AFF4 formed nuclear puncta in HCT 116 cells before and after treated with 1% 1,6-hexanediol (abbreviation as 1,6-hex) for 30 minutes. F Violin plot showing the number of cells containing nuclear puncta in Fig 1E. Results are representative of three biological replicates, each n > 20. Two-tailed, unpaired Student's t test was performed. For SPT5, p = 0.1301. For NELFA, p = 0.2756. For Pol II-S5P, p = 0.8179. For AFF4, ***p < 0.0001. GG-I ChIP-qPCR showing that the occupancies of Pol II (G), SPT5 (H), and NELFE (I) at the promoters and gene bodies of HSP70, GAPDH, and MYC in HeLa cells. The HEMO gene serves as a negative control for ChIP-qPCR. Error bars represent standard error of means. Results are plotted with four technical repeats from two biological replicates. Two-tailed, unpaired Student's t test was performed. For Pol II ChIP, HSP70_promoter, **p = 0.0014; GAPDH_promoter, p = 0.8385; GAPDH_gene body, ***p < 0.0001; MYC_promoter, p = 0.1765; MYC_gene body, ***p = 0.0002. For SPT5 ChIP, HSP70_promoter, **p = 0.0033; GAPDH_promoter, p = 0.9603; GAPDH_gene body, **p = 0.0012; MYC_promoter, p = 0.9898; MYC_gene body, ***p < 0.0001. For NELFE ChIP, HSP70_promoter, p = 0.3913; GAPDH_promoter, p = 0.9347; MYC_promoter, p = 0.8558.
Figure 2C Normalized FRAP curves for eGFP-SPT5. The bleaching events occurred at 0 second. Confocal images were embedded within the plot showing FRAP of the in vitro eGFP-SPT5 clusters. Results shown are from three biological replicates. The arrowhead represents the bleached region.E Live cell imaging of HEK-293T cells expressing eGFP-SPT5 together with mCherry-SPT5, mCherry-SPT5ΔNTR, or mCherry-SPT5ΔCTR close to endogenous level. F Violin plot showing the clusters area (top) and circularity (bottom) of SPT5 with SPT5, SPT5ΔNTR, or SPT5ΔCTR in Fig 2E. Each n > 20. Results are representative of three biological replicates, each n > 20. Two-tailed, unpaired Student's t test was performed. ***p < 0.001. GH Confocal images showing FRAP analysis of the NELFA-IDR-eGFP and SPT5-CTR-mCherry clusters in vitro. The arrowhead represents the bleached and recovered cluster. I Normalized FRAP curves for NELFA-IDR-eGFP and SPT5-CTR-mCherry clusters. The bleaching events occurred at 0 second. Results shown are from two biological replicates.
Figure 3A Western blot analysis of re-expression of mCherry-SPT5, mCherry-SPT5ΔNTR or mCherry-SPT5ΔCTR after SPT5 depletion. α-Tubulin was used as a loading control. Results are representative of two biological replicates.B Confocal imaging of nascent RNA labeled by EU in HEK-293T cells expressing SPT5, SPT5ΔNTR or SPT5ΔCTR after SPT5 depletion.G, H ChIP-qPCR showing the occupancies of Pol II at the promoters and gene bodies of GAPDH (G), and MYC (H) in HEK-293T cells expressing SPT5, SPT5ΔNTR or SPT5ΔCTR after SPT5 depletion. The HEMO gene serves as a negative control for ChIP-qPCR. Error bars represent standard error of means. Results are plotted with four technical repeats from two biological replicates. Two-tailed, unpaired Student's t test was performed. *p < 0.05, **p < 0.01, ***p < 0.001, n.s.=not significant. For GAPDH_promoter, p value (from left to right, < 0.0001, < 0.0001, < 0.0001, 0.0044, 0.8873, 0.9310, 0.6855, 0.9615); GAPDH_gene body, p value (from left to right, 0.0370, 0.0214, 0.0574, 0.0256, 0.9536, 0.8038, 0.7623, 0.8773); For MYC_promoter, p value (from left to right, 0.0002, 0.0014, 0.0004, 0.0048, 0.0670, 0.7501, 0.3482, 0.3284); MYC_gene body, p value (from left to right, 0.0004, 0.0013, 0.0081, 0.0299, 0.1332, 0.5839, 0.4404, 0.6170).
Figure 4B Fluorescence microscopy images showing the phase-separated AFF4-IDR-eGFP and SPT5-CTR-mCherry droplets formed in 50 mM NaCl containing buffer in the presence of 10% PEG-8000. The concentration ratios are indicated as CAFF4-IDR/CSPT5-CTR. C Dot plot showing the droplet area in Fig 4B. Fields per condition n = 5. DD Live cell confocal images showing FRAP of the heterotypic eGFP-AFF4 + ENL and mCherry-SPT5 droplets. The arrowhead represents the bleached and recovered droplet. E Normalized FRAP curves for eGFP-AFF4 + ENL (green) and mCherry-SPT5 (purple) in the AFF4/ENL/SPT5 heterotypic droplets. The bleaching events occurred at 0 second. Results shown are from three biological replicates.
Figure 5C Confocal imaging of FOS RNA FISH with concurrent SPT5 IF in wild type (WT) and ENL KO HCT116 cells after serum treatment. Zoomed-in views of the merged regions are indicated by the white arrow.H Confocal images showing SPT5 in nuclear puncta in WT or ENL KO cells treatment with KL-1. I Violin plot showing the number of cells containing nuclear puncta in Fig 5H. Results are representative of three biological replicates, each n > 20. Two-tailed, unpaired Student's t test was performed. ***p < 0.001.J-M (J) Confocal images showing co-localization of Pol II-S5P with SPT5 in nuclear puncta in KL-1 treated HCT 116 cells. Results shown are representative images from three independent experiments. (K) Boxplots showing the mean values of the Pearson correlation coefficient of co-localization ratios of SPT5 and Pol II-S5P in Fig 5J. Central band is median; boxes represent 1st and 3rd quartile (25th and 75th percentile, respectively) and whiskers 1.5× interquartile range. Results are representative of three biological replicates, each n > 20. Two-tailed, unpaired Student's t test was performed. KL-1 vs. DMSO, **p = 0.0050. (L) Same as Fig 5J. but for SPT5 and NELFA. (M) Same as Fig 5K. but for SPT5 and NELFA. Two-tailed, unpaired Student's t test was performed. KL-1 vs. DMSO, *p = 0.0100.
Figure 6B Fluorescence microscopy images showing that 2 μM SPT5-CTR-eGFP formed phase-separated droplets in the NaCl containing buffer. C Droplet number (top) and droplet area (bottom) analyses are shown. Central band is median; boxes represent 1st and 3rd quartile (25th and 75th percentile, respectively) and whiskers 1.5× interquartile range. Results are representative of two biological replicates, each n > 6. Two-tailed, unpaired Student's t test was performed. For droplet number, SPT5-CTR + P-TEFb (- ATP) vs. SPT5-CTR, p = **0.0015. SPT5-CTR + P-TEFb (+ vs.- ATP), **p = 0.0032. For droplet area, SPT5-CTR + P-TEFb (+ vs. - ATP)E Fluorescence microscopy images showing the phase-separated droplets formed in 50 mM NaCl containing buffer with 10 μM AFF4-IDR-eGFP and 10 μM SPT5-CTR-mCherry or SPT5-CTR-TE-mCherry in the presence of 10% PEG-8000. F Droplet number (top) and droplet area (bottom) analyses are shown. Central band is median; boxes represent 1st and 3rd quartile (25th and 75th percentile, respectively) and whiskers 1.5× interquartile range. Results are representative of two biological replicates, each n > 6. Two-tailed, unpaired Student's t test was performed. AFF4-IDR + SPT5-CTR-TE vs. AFF4-IDR + SPT5-CTR, **p = 0.0029.
Figure 7A Confocal images showing co-localization of AFF4 with SPT5 in nuclear puncta in AFF4 wild type (WT) or R258W cells. B Dotplot (left) showing the AFF4 or SPT5 puncta area in AFF4 WT or R258W cells (Fig 7A). Two-tailed, unpaired Student's t test was performed. AFF4 R258W vs. WT, *p = 0.0254. Violin plot (right) showing the Pearson correlation coefficient of co-localization ratio (Fig 7A). Each n > 20. Results are representative of three biological replicates. Two-tailed, unpaired Student's t test was performed. AFF4 R258W vs. WT, *p = 0.0166. CC Droplet experiments showing that CHOPS syndrome patient missense mutations reinforced AFF4-ALF domain droplet formation. D Dotplot showing the droplet areas in Fig 7C. Fields per condition n = 5. Two-tailed, unpaired Student's t test was performed. T254S, T254A or R258W vs. WT, *p < 0.05, **p < 0.01, ***p < 0.001. E AFF4-ALF-mCherry condensed fraction as a function of AFF4-ALF-mCherry concentration for experiments in Fig 7C. Error bars represent standard deviations. Fields per condition n = 5. Two-tailed, unpaired Student's t test was performed. T254S, T254A or R258W vs. WT, *p < 0.05, **p < 0.01. FK-N ChIP-qPCR showing the occupancies of Pol II (K), AFF4 (L), SPT5 (M), and PAF1 (N) at the promoter and gene body of HSP70 in AFF4 WT and R258W HEK-293T cells after heat shock for 2 hours. The HEMO gene serves as a negative control for ChIP-qPCR. Error bars represent standard error of means. Results are plotted with four technical repeats from two biological replicates. Two-tailed, unpaired Student's t test was performed. For Pol II ChIP, HSP70_promoter, ***p < 0.0001; HSP70_gene body, ***p = 0.0002. For AFF4 ChIP, HSP70_promoter, ***p < 0.0001; HSP70_gene body, ***p < 0.0001. For SPT5 ChIP, HSP70_promoter, ***p < 0.0001; HSP70_gene body, **p = 0.0086. For PAF1 ChIP, HSP70_promoter, ***p = 0.0007; HSP70_gene body, **p = 0.0021.
Figure 1Western blot analysis and quantitation of Sel1L-Hrd1 ERAD proteins in the livers of WT mice at 3, 9 and 24 weeks of age (n=3-6 per group, 2 independent repeats). α-tubulin, loading controls for Western blot analysis.Western blot analysis and quantitation of Sel1L-Hrd1 ERAD proteins in the livers of 10-week-old WT mice under fasted (overnight) or ad libitum fed conditions (n=3-6 per group, 2 independent repeats). α-tubulin, loading controls for Western blot analysis.Western blot analysis and quantitation of ERAD proteins in Sel1Lf/f and Sel1LAlb livers (n=4 per group, 3 independent repeats). Hsp90 loading controls for Western blot analysis.Growth curves of male (n=10 each) and female (n=7 each) mice.Representative images (E) and nose-to-anus length (F) of male mice at 6 weeks of age (n=6-10 per group).Organ-to-body-weight ratios of liver and kidney in 6-week-old male mice (n=6 per group, 3 independent repeats).Daily food intake (g/d) normalized to gram of body weight (gbw) (n=3 per group, measured over 3 days).Representative estrus cycle mapping in 2-4-month old females (n=6 per group).H&E images of paraffin-embedded liver sections from 9-week-old mice (n=4 per group, 3 independent repeats).Western blot analysis of cell death (cleaved and pro-caspase-3) in livers of 9-week-old mice, with WT ileum as positive control (n=3 per group, 3 independent repeats). Hsp90 loading controls for Western blot analysis.Representative transmission electron microscope (TEM) images obtained from 9-week-old female mice livers (n=10-12 cells from one mouse each). N, nucleus; mito, mitochondria; ER, endoplasmic reticulum.
Figure 2Volcano plot depicting transcriptomics data from the livers of 9-week-old Sel1Lf/f and Sel1LAlb mice (n=3 per group); dotted line marks p=0.05; black dots represent fold change>2.qPCR analyses of Fgf21 expression in 9-week-old livers (n=3-6 per group, 3 independent repeats). Ribosomal L32, loading control for qPCR analysis.Western blot analyses of Fgf21 expression in 9-week-old livers (n=3-6 per group, 3 independent repeats). α-tubulin, loading control for Western blot analysis.ELISA analysis of Fgf21 in serum from 8-9-week-old mice (n=6-7 per group).Protein levels of Sel1L (left panel) and mRNA levels of Fgf21 (right panel) in primary mouse hepatocytes isolated from the tamoxifen-inducible Sel1L-knockout Sel1LERCre mice (2 independent repeats). Hsp90 , loading control for Western blot analysis. Ribosomal L32, loading control for qPCR analysis.Acute loss-of-function model where 8-week-old Sel1Lf/f mice were injected i.v. with either AAV8-Cre or control AAV8-GFP: Western blot analysis of hepatic and control adipose Sel1L protein (n=3 per group); Hsp90 loading control for Western blot analysis.Acute loss-of-function model where 8-week-old Sel1Lf/f mice were injected i.v. with either AAV8-Cre or control AAV8-GFP: qPCR analysis of hepatic Fgf21 expression and ELISA analysis of serum Fgf21 (n=3 per group, 2 independent repeats). Ribosomal L32, loading control for qPCR analysis.Heatmaps of top 15 significantly upregulated and downregulated genes in Fgf21 Tg livers and their expression levels in Sel1LAlb livers (n=3 per group).Scatter plot depicting the logarithmic fold-change (FC) for 16,402 genes in Sel1LAlb and Fgf21 transgenic (Tg) livers (n=3 per group); genes that are highly upregulated or downregulated in both datasets are marked in red and blue, respectively; genes that are upregulated unique to each data set (e.g. Derl3 for Sel1L-Hrd1 ERAD-deficient liver) are marked in green.Data from rescue experiments where 5-week-old Sel1Lf/f and Sel1LAlb mice were injected i.v. with AAV8-shFgf21 or control AAV8-shLuc: qPCR analysis of Fgf21 mRNA 3 weeks after injection (n=4 per group). Ribosomal L32, loading control for qPCR analysis.Data from rescue experiments where 5-week-old Sel1Lf/f and Sel1LAlb mice were injected i.v. with AAV8-shFgf21 or control AAV8-shLuc: ELISA analysis of Fgf21 in serum (K) 3 weeks after injection (n=4 per group).Data from rescue experiments where 5-week-old Sel1Lf/f and Sel1LAlb mice were injected i.v. with AAV8-shFgf21 or control AAV8-shLuc: Weight gain curve after injection (n=7 per group).
Figure 3Western blot analysis of p-Stat5 in livers of 9-week-old mice (n=3 per group, 3 independent repeats), with quantitation of the ratio of p- to total Stat5 shown below the blot. Hsp90, loading control for Western blot analysis.qPCR analysis of p-Stat5-associated growth genes in the livers of 9-week-old mice (n=6 per group, 2 independent repeats). Ribosomal L32, loading control for qPCR analysis.Z ambulatory activity of 9-week-old male mice as measured over 24 hr (n=4 males per group).Blood glucose levels in 9-week-old mice after 6 hr fasting (n=6-10 per group).serum insulin levels in 9-week-old mice after 6 hr fasting (n=6-10 per group).serum triglyceride (TG) levels in 9-week-old mice after 6 hr fasting (n=6-10 per group).serum cholesterol (CHOL) levels in 9-week-old mice after 6 hr fasting (n=6-10 per group).H&E images of inguinal white adipose tissue (iWAT) from 8-week-old mice (n=3 per group, 2 independent repeats).qPCR analysis of browning-related genes in iWAT (n=3-6 per group). Ribosomal L32, loading control for qPCR analysis.Western blot analysis of Ucp1 in iWAT of 8-week-old mice (n=3-4 per group, 3 independent repeats). Hsp90, loading control for Western blot analysis.Weight gain curve of male mice after 60% high fat diet (HFD) starting at 5 weeks of age (n=4 per group, 2 independent repeats).Adipose tissue weight normalized to body weight in male mice following 9 weeks of HFD feeding (n=4 per group).
Figure 4Western blot analysis of Crebh, Crebh-N, Pparα and Xbp1s in whole cell lysates (A) and nuclear extracts (B) in WT and Sel1LAlb livers (n=3 per group, 3 independent repeats) with quantitation shown in (C). Hsp90 lamin, loading controls for Western blot analysis.qPCR analysis of Crebh, Ppara and Xbp1s in WT and Sel1LAlb livers (n=4-6 per group, 2 independent repeats). Ribosomal L32, loading control for qPCR analysis.Western blot analysis of Crebh in acute Sel1L loss of function model as described in Figure 2F-G (n=3 per group). Hsp90 , loading controls for Western blot analysis.Western blot analysis of Crebh protein in 9-week-old mice (n=3 per group, 2 independent repeats). WT mice injected i.p. with tunicamycin (Tm, 1.5 µg/g body weight) for 72 hours were included as a control. Hsp90 loading controls for Western blot analysis.Western blot analysis of Crebh in the livers of 9-week-old mice after NP40-detergent fractionation into NP40 soluble (NP40S) and pellet (NP40P) (n=3 per group, 2 independent repeats). Hsp90 H2A loading controls for Western blot analysis.Representative confocal images of Crebh in the liver cryosections of 8-week-old mice (zoomed out versions in Figure EV3F). Note that a fraction of hepatocytes is binucleated. Yellow arrows represent Crebh staining inside in the hepatocyte nucleus.ChIP analysis of Crebh binding onto the Fgf21 promoter in the livers of 9-week-old mice, normalized first to 5% input group and then to no-antibody ChIP samples (n=3 pooled per group, 2 independent repeats).Western blot analysis of SEL1L, HRD1 and CREBH proteins in human Hep3B hepatocytes upon CRISPR deletion of SEL1L with two different guides. , β-actin loading controls for Western blot analysis.qPCR analysis of SEL1L, HRD1 and FGF21 (K) in human Hep3B hepatocytes upon CRISPR deletion of SEL1L with two different guides. Ribosomal L32, loading control for qPCR analysis.
Figure 5Western blot analysis of Crebh protein half-life in transfected WT and HRD1-/- HEK293T cells treated with cycloheximide (CHX) for indicated times. The decay of protein from one experiment is shown below. Hsp90, loading control for Western blot analysis.Western blot analysis and quantitation of Crebh in Crebh-transfected WT (B) and HRD1-/- (C) HEK293T cells pre-treated with the proteasomal inhibitor bortezomib (BTZ) or lysosomal inhibitor chloroquine (CHQ) for 2 hours and then with CHX for additional 1 hour (n=2 per group, 2 independent repeats). Hsp90, loading control for Western blot analysis.Western blot analysis of Crebh ubiquitination following immunoprecipitation (IP) of Crebh-Flag and Crebh-N-Flag in HEK293T cells transfected with indicated plasmids. Samples were boiled with SDS before IP for denaturing IP and not so for native IP. These cells were treated with proteasomal inhibitor BTZ for the last 6 hr prior to immunoprecipitation. Hsp90, loading control for Western blot analysis.Western blot analysis of endoglycosidase H (endoH)-sensitivity of Crebh in the livers of 9-week-old mice; 'r' refers to endoH-cleavage-resistant species and 's' refers to endoH-cleavage-sensitive species (n=3 per group, 2 independent repeats). Hsp90, loading control for Western blot analysis.
Figure 65-week-old Sel1Lf/f and Sel1LAlb mice were injected i.v. once with AAV8-shCrebh or control AAV8-shLuc. Western blot analysis of hepatic Sel1L and Crebh 5 weeks post injection (n=3 mice each, 2 independent repeats). Hsp90, loading control for Western blot analysis.5-week-old Sel1Lf/f and Sel1LAlb mice were injected i.v. once with AAV8-shCrebh or control AAV8-shLuc. qPCR analysis of Crebh and Fgf21 mRNA 5 weeks post injection (n=3 per group, 2 independent repeats). Ribosomal L32, loading control for qPCR analysis.5-week-old Sel1Lf/f and Sel1LAlb mice were injected i.v. once with AAV8-shCrebh or control AAV8-shLuc. ELISA analysis of circulating Fgf21 (C) 5 weeks post injection (n=3 per group, 2 independent repeats).5-week-old Sel1Lf/f and Sel1LAlb mice were injected i.v. once with AAV8-shCrebh or control AAV8-shLuc. qPCR analysis of hepatic growth-associated genes 5 weeks after injection (n=6 per group). Ribosomal L32, loading control for qPCR analysis.5-week-old Sel1Lf/f and Sel1LAlb mice were injected i.v. once with AAV8-shCrebh or control AAV8-shLuc. Weight gain 6 weeks post injection (n=10 per group).5-week-old Sel1Lf/f and Sel1LAlb mice were injected i.v. once with AAV8-shCrebh or control AAV8-shLuc. Insulin tolerance test (ITT) 5-weeks after injection (n=10 per group).5-week-old Sel1Lf/f and Sel1LAlb mice were injected i.v. once with AAV8-shCrebh or control AAV8-shLuc. qPCR of Ucp1 levels in inguinal white adipose tissue (iWAT) 5 weeks post injection (n=3 per group, 2 independent repeats). Ribosomal L32, loading control for qPCR analysis.5-week-old Sel1Lf/f and Sel1LAlb mice were injected i.v. once with AAV8-shCrebh or control AAV8-shLuc. Western blot analysis (H) of Ucp1 levels in inguinal white adipose tissue (iWAT) 5 weeks post injection (n=3 per group, 2 independent repeats). Hsp90, loading control for Western blot analysis.
Figure 7Analysis of correlation between Sel1L-Hrd1 ERAD and Crebh-Fgf21 during fasting-feeding Western blot analysis of hepatic Sel1L-Hrd1 ERAD and Crebh of nuclear (Nuc) and cytosolic (Cyto) fractions from 10-week-old Sel1Lf/f and Sel1LAlb mice under overnight fasted or fed states.Analysis of correlation between Sel1L-Hrd1 ERAD and Crebh-Fgf21 during fasting-feeding Quantitation of levels of Sel1L/Hrd1/Crebh proteins and Fgf21 mRNA in the livers of 10-week-old Sel1Lf/f mice.during fasting-feeding Quantitation of total Crebh (Crebh+Crebh-N) protein levels in the livers of 10-week-old Sel1Lf/f and Sel1LAlb mice.during fasting-feeding Serum Fgf21 levels in 10-week-old Sel1Lf/f and Sel1LAlb mice.Analysis of correlation between Sel1L-Hrd1 ERAD and Crebh-Fgf21 during growth Western blot analysis of hepatic Sel1L-Hrd1 ERAD and Crebh of nuclear (Nuc) and cytosolic (Cyto) fractions from the livers from Sel1Lf/f and Sel1LAlb mice at 3, 9 and 24 weeks of age.Analysis of correlation between Sel1L-Hrd1 ERAD and Crebh-Fgf21 during growth Quantitation of levels of Sel1L/Hrd1/Crebh proteins and Fgf21 mRNA with the livers from Sel1Lf/f and Sel1LAlb mice at 3, 9 and 24 weeks of age.Quantitation of total Crebh (Crebh+Crebh-N) protein levels in the livers from Sel1Lf/f and Sel1LAlb mice at 3, 9 and 24 weeks of age.Serum Fgf21 levels in the livers from Sel1Lf/f and Sel1LAlb mice at 3, 9 and 24 weeks of age.
Figure 1(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 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 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-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.
Figure 2(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) Heatmap featuring the genes in MCODE Cluster 1.