Fig 2: GPR56 impairs cell migration through PI3K/AKT signaling. (A) The expression of p-AKT, AKT, PI3K and p-PI3K was evaluated by western blotting. (B) Western blotting and qRT-PCR of GPR56 protein and mRNA expression, respectively, in HT-29 NC and GPR56-overexpressing cells (GPR56) (*P<0.05). (C) Transwell migration assays revealed the relationship between GPR56, PI3K/AKT, and CRC cell migration. Magnification, ×100. (D) The number of migrated cells were counted (*P<0.05).

Fig 3: The association between EMT and PI3K/AKT signaling. (A) Western blotting detection of EMT-related target (E-cadherin, N-cadherin and vimentin) proteins and mRNA expression in HT-29 (NC), GPR56-overexpressing cells (GPR56), and GPR56 cells treated with the LY294002 inhibitor (GPR56 + LY294002). (B) qRT-PCR detection of EMT-related target proteins and mRNA (E-cadherin, N-cadherin and vimentin) expression in HT-29 NC and HT-29 with GPR56-overexpressing (GPR56), and GPR56 cells treated with LY294002 (GPR56 + LY294002) (*P<0.05).

Fig 4: GPR56 knockdown activates apoptosis of CRC cells. Flow cytometry was used to detect the apoptosis of (A) HCT116 and (B) DLD-1 cells (*P<0.05). (C) Western blotting and (D) qRT-PCR detection of protein and mRNA expression levels of Bax and Bcl-2 (*P<0.05). GAPDH was used as the control.

Fig 5: The effect of GPR56 on CRC cell proliferation. (A) The expression of GPR56 was detected by qRT-PCR in HCT116 and DLD-1 cell lines transfected with a negative control (si-NC) and siRNA targeting GPR56 (si-RNA1, si-RNA2, and si-RNA3) (*P<0.05). (B and C) A CCK-8 assay revealed that knockdown of GPR56 inhibited cell proliferation in (B) HCT116 and (C) DLD-1 cells (*P<0.05). (D and E) The effect of GPR56 knockdown on the HCT116 and DLD-1 cell cycle as assessed by flow cytometry.