Fig. 3
Knockdown of CCNB2 inhibits the malignant biological behaviors of GC cells. (A) Relative mRNA expression levels of CCNB2 in the normal gastric epithelial cell line GES-1 and three GC cell lines (HGC-27, AGS, and MKN-45), quantified by quantitative reverse transcription polymerase chain reaction (qRT-PCR). (B) Comparative analysis of protein expression levels of CCNB2 across the three GC cell lines and a human normal gastric epithelial cell line. (C-D) qRT-PCR (C) and Western blotting (D) were employed to establish stable transfection for both CCNB2 knockdown and overexpression in GC cells. (E) Wound healing assays were conducted to assess cellular migration capabilities; wound width was measured at 0 and 48 h, with the migration rate calculated at 48 h. n = 3.bar = 100 μm. (F) Transwell assays were performed to evaluate migratory activity following CCNB2 suppression, quantifying the number of cells migrating into the basal chamber after a 36-hour incubation period. n = 3. bar = 100 μm. (G) Flow cytometry was utilized to analyze alterations in the cell cycle distribution of GC cells post-CCNB2 suppression, categorizing cells into G1/G0 phase, G2/M phase, and S phase based on propidium iodide fluorescence intensity and DNA content. n = 3. bar = 100 μm. (H) The Cell Counting Kit-8 (CCK-8) proliferation assay assessed cellular growth at time points: 0, 24, 48, and 72 h at an absorbance wavelength of 450 nm; cellular proliferation capacity was evaluated accordingly. n = 3. (I) The colony formation assay determined the impact of CCNB2 downregulation on proliferative activity in GC cells; colonies were counted microscopically after conventional culture for ten days. n = 3. (J) Western blotting combined with ImageJ software analysis was employed to quantify expressions of CCNB2 as well as epithelial-mesenchymal transition-related proteins following downregulation of CCNB2 in these cells.Data are expressed as mean ± standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001.
