Fig. 5

Combination of Roblitinib and CPI-169 synergistically inhibits the YAP signaling. A Volcano Plot showed the differentially expressed genes between control group and CPI-169 + Roblitinib treatment group (fold change ≥ 2 or ≤  − 2 and p-values < 0.05). HepG2 cells were treated with CPI-169 + Roblitinib for 48 h, followed by RNA-Seq analysis. Red indicates high relative expression, and blue indicates low relative expression. B Bubble chart showed the KEGG pathway analysis of differentially expressed genes following CPI-169 + Roblitinib treatment in (A). P-values < 0.05 was regarded as statistically significant. C Gene set enrichment analysis (GSEA) showed that the Hippo signaling pathway was enriched in the co-treatment group compared with Control group. D Western bolt detected the expression level of YAP1 and p-YAP1 after CPI-169, Roblitinib or CPI-169 + Roblitinib treatment for 48 h. E Venn diagram showed YAP1 target genes that were regulated by CPI-169 + Roblitinib treatment. F Gene functional annotation of GO enrichment analysis of the 141 down-regulated YAP1 target genes in (E) via Metascape. G Heatmap representing the expression levels of 18 YAP1 target genes related to cell proliferation, cell migration and anti-apoptosis determined by RNA-Seq in HepG2 cells following CPI-169, Roblitinib or CPI-169 + Roblitinib treatment for 48 h. H Genomic tracks displayed ChIP-seq data for YAP1 around the indicated genes and their corresponding RNA-Seq signals in (G). I qPCR analysis of the representative YAP1 target genes from typical pathway related to cell proliferation, cell migration and anti-apoptosis in (G). Data are presented as mean ± SEM (n = 3, one-way ANOVA with Tukey’s multiple comparisons test, *P < 0.05, **P < 0.01, ***p < 0.001, ****p < 0.0001, ns, no significance). J Luciferase assay for YAP/TAZ activity in HepG2 and SMMC-7721 cells following CPI-169, Roblitinib or CPI-169 + Roblitinib treatment for 48 h. Data are presented as mean ± SEM (n = 3, two-way ANOVA with Sidak’s multiple comparison test, **P < 0.01, ****p < 0.0001)