Fig 2: GLI2 is required for cell invasion via augmentation of EMT in GBC. Invasion assay of GBC cell lines treated with (A) GLI2 siRNA or control siRNA, (B) GLI1 siRNA or control siRNA, and (C) GLI3 siRNA or control siRNA for 18 h. (D) Invasion assay of GBC cell lines treated with GLI2 siRNA or control siRNA and then incubated with or without rhSHH for 18 h. (E) Expression levels of GLI2 and EMT-related molecules (E-cadherin, vimentin, SNAI1, Slug, and TGF-ß1) in GBC cell lines treated with GLI2 siRNA or control siRNA. Scale bar, 100 µm. *P<0.05, **P<0.01 and ***P<0.001. Bar, mean ± SD. GLI2, glioma-associated oncogene homolog 2; EMT, epithelial-mesenchymal transition; GBC, gallbladder cancer; siRNA or si, small interfering RNA; siCont, control siRNA; siGLI2, GLI2 siRNA; siGLI1, GLI1 siRNA; siGLI3, GLI3 siRNA; rhSHH, recombinant human SHH; TGF-ß1, transforming growth factor ß1; n.s, not significant.

Fig 3: GLI2 is involved in cell proliferation in GBC via regulation of the cell cycle. (A) Expression levels of GLI3, GLI2, GLI1, and Hh signal-related molecules (SMO and SHH) were examined in three GBC cell lines by western blot analysis. (B-D) Proliferation assays in GBC cell lines treated with (B) GLI2 siRNA or control siRNA, (C) GLI1 siRNA or control siRNA, and (D) GLI3 siRNA or control siRNA for 24 and 48 h. (E) Proliferation assays in GBC cell lines treated with GLI2 siRNA or control siRNA and then incubated with or without rhSHH for 24 and 48 h. (F) Cell cycle analysis of GBC cell lines treated with GLI2 siRNA or control siRNA. Percentages of cells in the G0/G1, S, and G2/M phase were calculated. Histograms and the bar graphs are presented. (G) Expression levels of GLI2 and cell cycle-related molecules (cyclin D1, Ki-67, and cyclin B1) were examined in GBC cell lines treated with GLI2 siRNA or control siRNA. *P<0.05, **P<0.01 and ***P<0.001. Bar, mean ± SD. GLI2, glioma-associated oncogene homolog 2; GBC, gallbladder cancer; SMO, smoothened; SHH, sonic hedgehog; siRNA or si, small interfering RNA; rhSHH, recombinant human SHH; siCont, control siRNA; siGLI2, GLI2 siRNA; siGLI1, GLI1 siRNA; siGLI3, GLI3 siRNA; n.s, not significant.

Fig 4: GLI2 expression is inversely correlated to the number of tumor-infiltrating CD8-positive T lymphocytes and correlated to PD-L1 expression. Comparison of the number of tumor infiltrating (A) CD3-positive cells and (B) CD8-positive cells in GLI2-high and GLI2-low expression groups. Original magnification is ×400. (C) Comparison of tumor-infiltrating CD8-positive/FOXP3-positive cell ratio in the two groups. (D) Comparison of the number of tumor-infiltrating FOXP3-positive cells. Original magnification is ×400. (E) Comparison of the PD-L1-positive ratio in the two groups. (F) GLI2 and PD-L1 expression in GBC cell lines treated with GLI2 siRNA or control siRNA. (G) GLI2 expression of GBC cell lines incubated in normoxic or hypoxic condition for 48 h. (H) HIF-1a and GLI2 expression in GBC cell lines treated with HIF-1a siRNA or control siRNA in hypoxic conditions for 48 h. Scale bar, 50 µm. *P<0.05, **P<0.01 and ***P<0.001. GLI2, glioma-associated oncogene homolog 2; PD-L1, programmed cell death ligand 1; GBC, gallbladder cancer; HIF-1a, hypoxia-inducible factor 1a; FOXP3, forkhead box P3; siRNA or si, small interfering RNA; siCont, control siRNA; siGLI2, GLI2 siRNA; siHIF-1a, HIF-1a siRNA.

Fig 5: Schematic findings of the study. In GBC, GLI2 augmented the proliferative capacity and invasive capacity, and regulated tissue fibrosis and TILs in the TME. Hypoxia may be involved in the expression of GLI2. GBC, gallbladder cancer; GLI2, glioma-associated oncogene homolog 2; TILs, tumor-infiltrating lymphocytes; TEM, tumor microenvironment.