Fig. 4. Cortistatin-deficient HSCs show exacerbated fibrotic responses and excessive differentiation capacity to activated myofibroblasts. (A ) Nonparenchymal HSC-enriched fractions were isolated from wild-type (CST+/+ ) and cortistatin-deficient (CST-/- ) livers and cultured as described in the diagram. (B ) Analysis of glial fibrillary acidic protein (GFAP) and α-smooth muscle actin (αSMA) expression by immunofluorescence of HSC cultures to determine the percentage of non-activated HSCs (GFAP+/αSMA-), myofibroblasts GFAP+/αSMAlow and activated myofibroblasts αSMAhigh (with stress fibers) at different times (n=18 for 6d+1d cultures, n=5 for 6d+4d cultures). Representative images for each cell type used to the analysis are shown as examples. (C ) Expression of profibrogenic mediators in HSCs after primary cultures (n=3 cultures by duplicates). (D-G ) Gene expression profile in CST+/+ andCST-/- HSCs analyzed by next-generation RNA sequencing. (D ) Volcano plot depicting the differentially expressed genes (DEGs, with false discovery rate FDR-p<0.05) between CST-/- andCST+/+ HSCs (description for all DEGs is included in table S5). (E ) Unsupervised hierarchical clustering of the 640-top DEGs (with log2 fold-change >1 or <-1) separated the samples into two major groups. (F ) Fold-change expression of the top-25 upregulated DEGs in CST-/- HSCs. Red bars correspond to muscle-related genes (table S1 shows a detailed description of these DEGs). (G ) Gene ontology (GO) analysis of the DEGs betweenCST-/- and CST+/+HSCs. The top-10 or top-5 terms are included. Red and blue bar-segments correspond to numbers of upregulated and downregulated DEGs, respectively. All DEGs in each GO term are listed in tables S2 to S4. (H ) Effect of cortistatin addition every other day to primary cultures of CST-/- HSCs on myofibroblast activation/differentiation (immunofluorescence at day 4, n=11 cultures) and expression of critical genes for myofibroblast activation and muscle differentiation (n=3 cultures by duplicates). Data are the mean±SEM (unless data in F that are the mean). *p<0.05, **p<0.01, ***p<0.001, unpaired two-tailed Student’s t-test.