The RhoA-F-actin axis mediated the signalling between GPR4 and the hippo pathway.
(a) Immunofluorescence (IF) staining analysis of F-actin labelled with TRITC phalloidin in GPR4-depleted HCT116 cells or control cells. Scale bars, 20 μm. (b) Representative western blot quantification of the G-actin/F-actin ratio in GPR4-depleted HCT116 cells or control cells. (c) Western blot quantification the level of GPR4, activated RhoA, p-LMIK, p-cofilin, p-LATS1 and p-YAP1 in GPR4-depleted HCT116 and HT29 cells. (d) Western blot quantification of the level of GPR4, activated RhoA, p-LMIK, p-cofilin, p-LATS1 and p-YAP1 in SW620 and SW480 cells with GPR4 overexpression and RhoA inhibition by COG1432 and Rhosin or the corresponding control cells. (e) Luciferase report assay of the CTGF promotor in HCT116 cells with the indicated treatment. (f) 3D on-top growth assay of SW480 and SW620 cells with GPR4 overexpression or GPR4 overexpression plus COG-1432. Scale bars, 100 μm. Differences between the groups in (b, e) were analysed by unpaired Student's t-test (* P < .05; ** P < .01; *** P < .001).