Reduced JAK2-STAT3 phosphorylation in Notch1-deficient hepatocytes. (A) The total RNA was isolated from liver of Notch1^fl/fl mice or HC Notch1^−/− mice and subjected into RNA-sequencing analysis. Differential gene expression between Notch1^fl/fl and HC Notch1^−/− group was analyzed and presented as volcano map. The x-axis shows the fold change, and the y-axis shows the P value. Red dots indicated the differentially upregulated genes with significance, and green dots presented the differentially downregulated genes with significance. (B) Signaling pathways enriched from KEGG pathway–based analysis. (C) Hepatocyte was isolated from Notch1^fl/fl and HC Notch1^−/− mice to measure the phosphorylation of JAK2/STAT3 and TPO level by western blot (mean ± standard deviation [SD]; n = 3; unpaired student t test). (D) Cultured hepatocytes were incubated with desialylated platelets for 30 minutes or 60 minutes, followed by measuring the phosphorylation of JAK2/STAT3, TPO level, Notch1 activation (cleaved Notch1 expression), and HES5 expression by western blot (mean ± SD; n = 3; 2-way ANOVA). (E) Expression of HES1 and HES5 in the hepatocyte of Notch1^fl/fl and HC Notch1^−/− mice was detected by western blot (mean ± SD; n = 3; unpaired student t test). The phosphorylation level of JAK2 and STAT3 was quantified as a ratio relative to respective total level, and the expression of TPO, HES5, HES1, and cleaved Notch1 was quantified as a ratio relative to β-actin level. ∗∗P < .01; ∗∗∗P < .001. ABC, ATP-binding cassette; PPAR, peroxisome proliferator-activated receptor; TGF-β, transforming growth factor-β.