Fig. 4

FOS perturbation boosts hepatocyte differentiation and maturation in ICOs. a Flowchart of individual validation of Fos perturbation in ICOs. b Western blot analysis of Fos protein levels in mICOs transduced with NT sgRNA or sgRNA targeting Fos. Uncropped images of the Western blots are available in Additional File 1: Fig. S12. c Volcano plot of differentially expressed genes (|log2FoldChange|< 1 and an adjusted P-value using the Benjamini-Hochberg (BH) method < 0.05) in Fos KO mICOs compared with NT control ICOs in DM condition. d The top 10 enriched GO-BP terms (sorted by adjusted P-values using the BH method) of significantly upregulated genes in Fos KO mICOs compared with the NT controls in DM condition. e, f Heatmap showing relative expression of genes involved in xenobiotic metabolism (e) or lipid metabolism (f). g Representative immunofluorescent staining of Alb expression in Fos KO mICOs compared with NT control ICOs in DM condition. Scale bars, 100 μm. h Percentages of Albumin positive cells for mICOs in g were shown as mean ± s.e.m. (n = 5) and compared by two-tailed Student’s t test. A P-value less than 0.05 was considered statistically significant. i, j Secretory Alb protein levels (i) and bile acid production (j) were measured in Fos KO mICOs compared with the NT controls in DM condition. Data were shown as mean ± s.e.m. (n = 6). Following one-way ANOVA, the Bonferroni method was used as the post hoc test for pairwise comparisons. Statistical significance was defined as a P-value below 0.05. k The expression dynamics of Fos, Hnf4α, and 20 reported TFs upregulated during hepatocyte development [43] using the previously published single-cell RNA-seq data [8]. l qPCR analysis showing the expression of hepatocyte genes in human FOS KO ICOs compared with the NT controls in DM conditions. m ALB protein levels as measured by immunofluorescent staining in human FOS KO ICOs compared with the NT controls in DM conditions. Scale bars, 50 μm