Extended Data Fig. 3: Bmp8b is overexpressed by Primary Hepatic Stellate Cells when cultured in vitro.

Relative mRNA gene expression levels measured by RNA sequencing (a-c) or quantitative real-time polymerase chain reaction (RTqPCR; d) in murine primary hepatic stellate cells cultured at a density of 35,000 cells/cm² and harvested before (day 0) and after culture (days 1, 4, 8, 12). Average mRNA abundance (Log2CPM) of BMP/TGFβ receptors and effectors (a), and of TGFβ/BMP family members (b): Artn, Bmp 2/8a, and Inhb b/c/e were suppressed; Bmp3/5/7/9/10 and Gdf9/10 were suppressed after a transient upregulation at day 1 of culture; Tgfβ2/3, Gdf6 and Inhba were upregulated (Nodal and Gdf3 were not expressed by HSC). c, IPA “upstream regulator” analysis based on time-dependent GE changes (NGS) in HSC at different stages of the trans-activation program compared to Day 1 (D1) of culturing shows activation of multiple TGFβ-related effectors Detailed NGS analysis (significantly modulated genes, IPA analysis) available in Supplementary Table 5; statistical significance was assessed by GLM likelihood ratio (edgeR) and then adjusted by the Benjamini-Hochberg procedure to control the False Discovery Rate (FDR). “Upstream Regulators” shown are all significantly enriched (P<0.05 - and with a 2≤Z-Score≥2) in at least one comparison. d, Time-dependent changes of Bmp8b expression in HSC treated with drivers of HSC activation (PDGFB 10ng/mL; Oleic Acid 100 μM; Palmitic Acid 100 μM; TNFα 30ng/mL; LPS 50 ng/mL). Bmp8b mRNA expression levels are very low at baseline and induced 24h after culturing (Area Under the Curve, AUC, in the small panel); Palmitic Acid significantly induces Bmp8b, while LPS reduces its expression over the time. All the results are shown as mean ± s.e.m. or in a heatmap format (representing gene abundance expressed as Log2CPM, or the degree of “Upstream Regulator” activation -Z-score- at the IPA “Upstream Regulator” Analysis). Sample Size: 4 biological replicates/group for panels a-c (each biological replicate is a pool of 3 livers); 3 replicates/time point for panel (d). Statistical significance was assessed by one-way analysis of variance (ANOVA) plus Fisher’s least significant difference test (d). Lowercase letters indicate post hoc analysis significance: a, reference group; groups with different letters are statistically different per post hoc comparison; differences between groups with the same letter are statistically not significant per post hoc comparison.