Fig. 2: USP12 depletion inhibits cell proliferation and promotes cell apoptosis in gastric cancer cells.

A Western blotting demonstrated knockdown efficiency of USP12. AGS and MKN74 cells transfected with 50 nM siControl or two independent siUSP12. β-Actin was used as the internal control. B, C USP12 depletion inhibits the proliferation of gastric cancer cells. AGS and MKN74 cells were transfected with 50 nM siControl or two independent siUSP12. After 24 h, CCK-8 assay was used to determine cell absorbance at the indicated time points after transfection. Experiments were performed in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001 for cell growth comparisons. D–G USP12 depletion reduced the number of migration and invasion gastric cancer cells. AGS and MKN74 cells were transfected with 50 nM siControl or two independent siUSP12. After 24 h, siControl and siUSP12 cells were separately plated into the Transwell chambers. For invasion experiments, it is necessary to spread the matrix gel before planting the cells. After 12 h, the cells were fixed and stained with crystal violet. The transwell cell number was determined to indicate cell migration (upper) and invasion (lower) activity (D, F). Quantification of transwell results using ImageJ software (E, G). Scale bar 100 μm. N = 3, *P < 0.05; **P < 0.01; ***P < 0.001 for cell migration comparisons. H–K Cell-cycle analysis by flow cytometry of AGS and MKN74 cells transfected with 50 nM siControl or two independent siUSP12. After 24 h, the cells were harvested, fixed with 70% ethanol, and stained with propidium iodide. The cells were subjected to FACS analysis (H, I). Quantitative summary of cell-cycle analysis using graphpad software (J, K). Experiments were performed in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001 for cell proportion comparisons. L–O Representative plots of apoptosis AGS and MKN74 cells transfected with 50 nM siControl or two independent siUSP12. After 24 h, the cells were harvested, and stained with Annexin V-PI (L, M). Quantitative summary of apoptosis analysis using graphpad software (N, O). Experiments were performed in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001 for cell proportion comparisons. P–R USP12 depletion inhibits the tumor growth of AGS cells in the xenograft model. Harvested and photographed tumors in the shUSP12 and shControl (P), tumor volume (Q) and weight (R) growth in each mouse from the shUSP12 and the shControl in vivo. *P < 0.05; **P < 0.01; ***P < 0.001.