Fig 2: Effects of GRHL3 on tumorigenicity of CRC cells in vivo. (a) Tumor volumes of different groups. *P < 0.05, **P < 0.01 vs. mouse inoculated cells transfected with NC. (b) The HE staining of the tumor.

Fig 3: Decreased GRHL3 expression represses cell proliferation and invasion. (a) The protein expression of ß-catenin, ERK1/2, p-ERK1/2, Vimentin, and E-cadherin was assayed by western blotting. (b) Cytoskeletal staining of F-actin. (c-e) Cell migration and invasion were detected by transwell in different treatment cells. (f, g) Vimentin and E-cadherin were assayed using immunofluorescence staining. Stably transfected knockdown expression of GRHL3 (GRHL3-KD) and negative control (NC) HT29 cells was treated with U0126 or solvent (vehicle).

Fig 4: Knockdown of GRHL3 inhibits EMT mediated by the MAPK/ERK pathway. (a) qPCR results showed the mRNA level of GRHL3 in HT29, SW480, HCT116, and SW620 cell lines. The data are presented as mean ± SEM (n = 3). (b) HT29 cells were transfected with three shRNA targets GRHL3 (KD-1, KD-2, and KD-3) or a scrambled sequence. The interference efficiency of GRHL3 expression was measured using qPCR. **P < 0.01 vs. negative control (NC). (c) In vitro cell growth of HT29 stably transfected cells was examined by the cell proliferation assay. **P < 0.01 compared with cells transfected with NC. (d) Cell migration was detected by transwell in two stable cells. (e) Protein expression of ß-catenin, p-ERK1/2, ERK1/2, E-cadherin, and Vimentin was detected using western blotting, and ß-tubulin served as a loading control.

Fig 5: Protein and mRNA expression of GRHL3 was analyzed in CRC. (a) Immunostaining of the CRC tumor and the adjacent nontumorous tissue. (b) qPCR analysis of the mRNA expression of GRHL3 in the tumor (n = 30) and the adjacent nontumorous tissue (n = 20) from CRC patients and ß-actin as the loading control. Data are presented as mean ± SEM. **P < 0.01 vs. the adjacent nontumorous tissues.