Fig. 5: YAP rescues gastric cancer cell suppression induced by USP12 deletion.

A Western blotting analysis showing USP12 depletion decreases YAP protein stability could be rescued by overexpression of YAP. AGS cells transfected with 50 nM siControl or siUSP12, and after 24 h, transfected with 0.5 μg Myc or Myc-YAP. Cell lysates were immunoblotted with the indicated antibodies. β-Actin was used as internal control. B qRT-PCR analysis of YAP target genes (CTGF, CYR61) expression. AGS cells transfected with 50 nM siControl or siUSP12 for 48 h, and after 24 h, transfected with 0.5 μg Myc or Myc-YAP. Total RNA was extracted for gene expression analysis. Each group was analyzed in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001 for target gene expression comparison. C Luciferase assays showing USP12 depletion affects TEAD-luciferase activity and its could be rescued by YAP expression. Each group was analyzed in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001 for luciferase activity comparison. D USP12 depletion inhibits the proliferation of gastric cancer cells could be rescued by overexpression of YAP. AGS cells transfected with 50 nM siControl or siUSP12 for 48 h, and after 24 h, transfected with 0.5 μg Myc or Myc-YAP. 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. E, F USP12 depletion inhibits the migration of gastric cancer cells could be rescued by overexpression of YAP. AGS cells transfected with 50 nM siControl or siUSP12 for 48 h, and after 24 h, transfected with 0.5 μg Myc or Myc-YAP. 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 (E). Quantification of transwell results using ImageJ software (F). Scale bar 100 μm. N = 3, *P < 0.05; **P < 0.01; ***P < 0.001 for cell migration comparisons. G, H USP12 depletion inhibits cell cycle progression, and it could be rescued by overexpression of YAP. AGS cells transfected with 50 nM siControl or siUSP12 for 48 h, and after 24 h, transfected with 0.5 μg Myc or Myc-YAP. After 24 h, the cells were harvested, fixed with 70% ethanol, and stained with propidium iodide. The cells were subjected to FACS analysis (G). Quantitative summary of cell-cycle analysis using graphpad software (H). Experiments were performed in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001 for cell proportion comparisons. I, J USP12 depletion inhibits cell apoptosis, and it could be rescued by overexpression of YAP. AGS cells transfected with 50 nM siControl or siUSP12 for 48 h, and after 24 h, transfected with 0.5 μg Myc or Myc-YAP. After 24 h, the cells were harvested, and stained with Annexin V-PI (I). Quantitative summary of apoptosis analysis using graphpad software (J). Experiments were performed in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001 for cell proportion comparisons. K–M USP12 depletion inhibits the tumor growth of AGS cells in the xenograft model, it could be rescued by overexpression of YAP. Harvested and photographed tumors in the shControl, shUSP12 and shUSP12+YAP (K), tumor volume (L) and weight (M) growth in each mouse. *P < 0.05; **P < 0.01; ***P < 0.001.