Fig. 5. RICH1 activates the kinases cascade of Hippo signaling and inhibits nuclear translocation of YAP/TAZ.
A The protein-protein interaction network between RICH1 and main regulators and effectors of Hippo signaling identified by STRING database. ARHGAP17-gene symbol for RICH1; NF2-gene symbol for MERLIN. B Detection of main regulators and effectors of Hippo signaling after RICH1 knockdown and overexpression by Western blotting. C, D The mRNA expression levels of YAP/TAZ target genes CTGF (C) and CYR61 (D) assayed by real time PCR. E TEAD-luciferase reporter analysis in RICH1-knockdown and -overexpressing cells. F, G Localization of YAP and TAZ shown by immunofluorescence assays. H Cytoplasmic and nuclear expression of YAP and TAZ determined by Western blotting. GAPDH was used as the loading control for the cytoplasmic protein. Lamin A/C was used as the loading control for nuclear protein. I Quantitation of ALDHhi subpopulations in BT549 with RICH1 overexpression and TAZ4SA transduction. J Quantitation of primary and secondary tumorsphere formation in BT549 cells with RICH1 overexpression and TAZ4SA transduction. K Relative mRNA expression levels of CSC-TFs (OCT4, SOX2, and NANOG) in BT549 cells with RICH1 overexpression and TAZ4SA transduction. All the experiments were repeated three times independently with similar results. Data represent mean ± SD. Statistical significance was determined by two-tailed unpaired t-test. Abbreviation: ns, not significant; **p < 0.01; ***p < 0.001.