Fig. 4. STK40 and MAPK signaling regulated YAP-mediated FA remodeling in collaborative manners.

(A) Top: Diagram of FL STK40, STK40 with KD only, and ΔKD constructs expressed in SAS cells. Bottom: Quantification of integrated FA intensity in SAS cells overexpressing above vectors. YFP, control vector. (B) Representative images of FAs of (A). (C) Top: Diagram of STK40-EYFP and STK40-EYFP-NES constructs expressed in SAS cells. Bottom: Quantification of integrated FA intensity in SAS cells overexpressing above vectors. (D) Representative images of FA of (C). 4of (Diamidino-2-phenylindole (DAPI) channel revealed locations of cell nuclei. YFP channel denotes subcellular distribution of above vectors. Note the distinct cytosolic translocation of STK40 by the NES. (E) Representative images and quantification of FA in SAS cells without or with shSTK40 treated with 100 nM trametinib overnight. Note that the trametinib effect was masked by STK40 knockdown in high efficiency. (F) Representative images of α-SMA in fibroblast HS68 cells without or with shSTK40 treated with 100 nM trametinib overnight. Note that the lower knockdown efficiency of STK40 in HS68 cells than in SAS cells resulted in marked synergistic enrichment of α-SMA on stress fibers of HS68 cells under combination treatment. (G) YAP activity [labeled by connective tissue growth factor (CTGF) expression] was decreased in SAS cells treated with (top) verteporfin (VP) and (bottom) trametinib, shRNA targeting cyclic adenosine 3′,5′-monophosphate response element–binding protein (shCREB), or shSTK40. HPRT, hypoxanthine-guanine phosphoribosyltransferase. (H) Representative images and quantification of FA in SAS cells without or with VP treated with shCTRL or shSTK40. (I) Representative images and quantification of FA in SAS cells with shSTK40 and treated with VP and/or trametinib. Note that shSTK40 and VP abolished effects of trametinib. Error bars denote means ± SEM. *P < 0.05. ns, not significant.