Fig. 2

AnxA6 inhibits HCC cell migration via weakening RHOU/AKT1-involved EMT. (A) Ectopic expression of AnxA6 was confirmed to interact with GFP-tagging RHOU protein. Co-transfection with plasmids pEGFP-RHOU and pFlag-AnxA6 into HepG2 cells, then Flag-tagging AnxA6 was enriched by co-IP, and the AnxA6-interacting RHOU was detected in protein precipitation using anti-GFP antibody. (B) Ectopic expression of AnxA6 was confirmed to interact with endogenous RHOU protein. HepG2 cells were transfected with pFlag-AnxA6 plasmids for 48 h, the lysates were performed IP to capture AnxA6 and subsequently immunoblotted with RHOU antibody. (C) Endogenous AnxA6 interacts with RHOU. The lysates of HepG2 cells were performed IP to capture endogenous AnxA6 and subsequently immunoblotted with RHOU antibody. (D) Protein expression level and quantification analysis of EMT markers and p-AKT1^ser473 in HEK293T and HepG2 cells. HEK293T and HepG2 cells were transiently transfected with pEGFP-RHOU for 48 h, and then several key EMT markers and p-AKT1^ser473 were detected. (E) AnxA6 overexpression inhibited cell migration via retarding RHOU-AKT1 signaling pathway. Plasmid pEGFP-RHOU was transiently transfected with or without pFlag-AnxA6 into HepG2 cells for 48 h, and EMT markers and p-AKT1^ser473 were detected in the indicated groups (left). Data were represented as the mean ± SD of three separate experiments (right). ns, no statistical; *P < 0.05; **P < 0.01; ***P < 0.001. (F) HepG2 cells were transfected with pFlag-AnxA6 plasmids and treated with 5μM AKT activator SC79 for 24 h to measure EMT markers. (G) HepG2 cells were transiently transfected pFlag-AnxA6 with pEGFP-RHOU for 48 h and treated with 5μM AKT activator SC79, then several key EMT markers and p-AKT1^ser473 were detected. (H) HepG2 cells were transfected with pFlag-AnxA6 plasmids and treated with 10μM inhibitor MK2206 for 24 h to measure EMT markers