(A) Immunoblots of total lysates of control, and GCKIII (STK25, MST3 and MST4) knockout (KO) 293A cells with the indicated antibodies. Anti-GAPDH blot was used as the loading control. (B) Immunofluorescence staining of YAP localization in control and the indicated GCKIII KO 293A cells. Cells were fixed, permeabilized, and stained with anti-YAP (green) and DAPI (blue). Scale bar, 5 μm. (C) Quantification of immunofluorescence signal intensities in (B). Approximately 100 cells were counted from five random fields of each sample. N < C (blue), N = C (grey), and N > C (red) categories indicate YAP localization in cytoplasm, both cytoplasm and nucleus, and nucleus, respectively. Data are plotted as mean ± SEM of three biological replicates. (D) Quantitative RT-PCR. Relative mRNA expression of YAP target genes CTGF and CYR61 in control and the indicated GCKIII kinase KO 293A cells. Data are plotted as mean ± SEM of three biological replicates (*p<0.05; ****p<0.0001; ns, non-significant). (E) Cell proliferation assay was performed in 293A cells. Cell proliferation curves in control (black), STK25 KO (purple), and STK25_MST1/2 TKO (orange) cells were plotted, respectively. Cells were counted on days 2, 4, and 6 after seeding. Data shown are the means ± SEM of three independent experiments. Numbers of STK25 KO or STK25_MST1/2 TKO cells on day 6 was compared to that of control cells (*p<0.05; ***p<0.001). (F) Immunoblots of control, STK25 KO, and STK25_MST1/2 TKO 293A cell lysates with the indicated antibodies. (G) Relative mRNA expression of YAP target genes CTGF and CYR61 in control, STK25 KO, and STK25_MST1/2 TKO 293A cells. Data are plotted as mean ± SEM of three biological replicates (**p<0.01; ***p<0.001).