Fig. 4: STAT1 stimulates YAP1 nuclear function in mutant KRAS cells.
a HCT116 and HK2-8 cells replete or deplete of STAT1 were serum-starved for 18 h (untreated; UT) and stimulated with either 10% fetal bovine serum or 25 μM LPA for 1 h. Cells were subjected to IF analyses of YAP1 (red) along with DAPI staining of DNA (blue). Graphs show the quantification of YAP1 nuclear localization in 300 cells. Scale bar: 25 μm. b, c Cells were subjected to cytoplasmic (C), and nuclear (N) fractionation followed by immunoblotting for the indicated proteins. TUBULIN or THO complex 1 (THOC1) was used as cytoplasmic or nuclear marker, respectively. Quantification in panel b is based on three biological replicates, while panel c is based on two biological replicates. d, e HCT116 STAT1+/+ and STAT1−/− cells were transfected with either pGL3-luciferase reporter plasmid (control) or 8xGTIIC plasmid containing the firefly luciferase reporter gene under the control of 8x TEAD binding sites in CTGF minimal promoter. Transfected cells were serum-starved for 18 h followed by stimulation with either 10% fetal bovine serum or 25 μM LPA for 6 h. A plasmid expressing the renilla luciferase gene was used as internal control. f, g HCT116 cells were serum starved for 18 h followed by stimulation of 10% fetal bovine serum in the absence or presence of 2.5 mM cerivastatin (panel f, g) or 10 μM ROCK kinase inhibitor Y-27632 (panel f) for 18 h. Protein extracts were subjected to immunoblotting for the indicated proteins. a, b, d, e Graphs show the quantifications from 3 biological replicates and represent ±SEM *P < 0.05, **P < 0.01, ***P < 0.001 (t-test), NS, non-significant. In c, data represent the quantification of 2 biological replicates.
