Fig. 3
ECZeb2KO and WD-exposure both induce LSEC proliferation early during MASLD. (a) Schematic diagram of the RNAseq set-up (left), overview of (3) pairwise comparisons (middle) and quantification of the number of differentially expressed genes (DEGs) per comparison (right) at 4 weeks (w) of standard diet (SD) or western-type diet (WD). A red dotted line separates the up-and downregulated number of DEGs (total number is indicated on top of bars). Combi: combined challenge. (b) Venn diagram (middle) showing overlap (205 DEGs with expression changes in the same direction) between DEGs from the knockout (KO; comparison 1 in purple) and the diet effect (comparison 2 in green) and expression of representative DEGs (normalized data; corresponding to functional terms highlighted in red in panel (c); n = 5–6) extracted from comparison 1 (top) and 2 (bottom). (c) Functional annotation analysis on the combined up- and downregulated DEGs. Bubble plots represent the top 10 functional terms (biological processes on the left, pathways on the right) ranked according to false discovery rate (FDR) related to comparison 1 (top), comparison 2 (bottom) and the overlap between comparison 1 and 2 (middle). Significance level in (c) indicated by red dotted lines. All quantitative data are also shown in Supplementary Table 1. (d-h) Liver cross-sections of ECGFP (d,f) or ECZeb2KO (e,g) mice after 4w of SD (d,e) or WD (f,g) co-stained for eGFP (green) and KI67 (red) and corresponding quantification (h) of proliferating cells (indicated by white arrowheads) expressed as % KI67+ cells (n = 4–6). Single-color images of the delineated inset are shown below each panel. Quantitative data are expressed as mean ± s.e.m; Panel (b): *: FDR (false discovery rate) < 0.05 vs. indicated condition by differential gene expression analysis with applying negative binomial general linear model approach and Benjamini-Hochberg multiple testing correction. Panel (h) *: P < 0.05 by two-way ANOVA with Tukey post-hoc test. Pictures in (d–g) were taken with a Plan-Apochromat 40x/1.3 Oil DIC M27 objective on a Zeiss LSM 700 AxioObserver Z1 equipped with an LSM T-PMT detector and ZEN software.
