Fig. 3: USP12^WT but not USP12^C48S promotes cell proliferation and inhibits cell apoptosis in gastric cancer cells.

A Schematic structure of the USP12 wild-type (Flag-USP12^WT) and enzyme inactivation mutant (Flag-USP12^C48S). B Western blotting demonstrated overexpression of USP12^WT and USP12^C48S. AGS cells transfected with 1 μg Flag or Flag-USP12^WT/Flag-USP12^C48S. β-Actin was used as the internal control. C USP12^WT but not USP12^C48S promotes cell proliferation. AGS cells were transfected with 1 μg Flag or Flag-USP12^WT/Flag-USP12^C48S. 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, E USP12^WT but not USP12^C48S promotes cell migration. AGS cells were transfected with 1 μg Flag or Flag-USP12^WT/Flag-USP12^C48S. After 24 h, 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). Quantification of transwell results using ImageJ software (E). Scale bar 100 μm. N = 3, *P < 0.05; **P < 0.01; ***P < 0.001 for cell migration comparisons. F, G USP12^WT but not USP12^C48S promotes cell cycle progression. AGS cells were transfected with 1 μg Flag or Flag-USP12^WT/Flag-USP12^C48S. After 24 h, the cells were harvested, fixed with 70% ethanol, and stained with propidium iodide. The cells were subjected to FACS analysis (F). Quantitative summary of cell-cycle analysis using graphpad software (G). Experiments were performed in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001 for cell proportion comparisons. H, I Representative plots of apoptosis AGS cells transfected with 1 μg Flag or Flag-USP12^WT/Flag-USP12^C48S. After 24 h, the cells were harvested, and stained with Annexin V-PI (H). Quantitative summary of apoptosis analysis using graphpad software (I). Experiments were performed in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001 for cell proportion comparisons.