Fig. 3

H. pylori triggers malignancy in MKN45 cells via regulating the GNB4 expression. A, B Gene set enrichment analysis (GSEA) based on gene expression profiling of patients with GC (n = 433) in the GSE84437. GNB4 significantly correlates with cell adhesion molecules (CAMs) (A) and ECM-receptor interaction pathway (B). C–E CCK-8 assay was performed to monitor the cell proliferation in MKN45 shControl or shGNB4 (C) and changes in proliferation of MKN45 shControl (D) and shGNB4 (E) uninfected or infected with H. pylori strains (26695 and SS1; 6 h). F, G Colony formation assays were performed to evaluate the proliferation abilities of MKN45 shControl and shGNB4 uninfected or infected with H. pylori strains (26695 and SS1; 6 h). Representative images (F) and histograms presenting the colony numbers in each group (G) are shown. H, I EdU assays were conducted in MKN45 shControl and shGNB4 uninfected or infected with H. pylori strains (26695 and SS1; 6 h) to compare the percentage of cells in the S phase (scale bar: 100 μm). DAPI staining detected total cells, whereas EdU staining represented cells with active DNA replication. Representative images (H) and quantification data (I) are shown. J, K. Wound healing assays were conducted to compare the migration capabilities of MKN45 shControl and shGNB4 uninfected or infected with H. pylori strains (26695 and SS1; 6 h). The difference in cell margin at 0 and 72 h showed the moving track of cells (scale bar: 200 μm) (J), and the percentage of the healed area was quantified (K). L–N Transwell assays of MKN45 shControl and shGNB4 uninfected or infected with H. pylori strains (26695 and SS1; 6 h) were performed to measure the migration and invasion abilities of cells (scale bars: 100 μm). Bar charts show the number of cells which passed through the chamber membrane in each group (M, N). Data are presented as the mean ± SD of three independent experiments. O Protein expression of GNB4, epithelial marker E-cadherin, mesenchymal markers (N-cadherin and vimentin), and survivin in control and shGNB4-expressing GC cells