Fig. 4

DUB1 controls gastric cancer progression via the Hippo/TAZ axis A: DUB1 depletion decreased the TAZ protein level, and this effect was reversed by TAZ overexpression. MGC803 cells were transfected with siControl or siDUB1. After 24 h, the cells were transfected with Flag-TAZ or siControl. After 48 h, the cells were harvested for western blot analysis. DUB1 and TAZ protein levels were determined by Western blotting. Actin was used as the internal control. B: DUB1 depletion decreased the TAZ protein level, and this effect was reversed by TAZ overexpression. AGS cells were transfected with siControl or siDUB1. After 24 h, the cells were transfected with Flag-TAZ or siControl. After 48 h, the cells were harvested for western blot analysis. DUB1 and TAZ protein levels were determined by Western blotting. Actin was used as the internal control. C: DUB1 depletion suppressed Hippo target gene expression, and this effect was reversed by TAZ overexpression. MGC803 cells were transfected with siControl or siDUB1. After 24 h, the cells were transfected with the Flag-TAZ or Flag vector. After 48 h, total RNA was extracted for gene expression analysis. Each group was tested in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001 for comparisons of target gene expression. D: DUB1 depletion suppressed Hippo target gene expression, and this effect was reversed by TAZ overexpression. AGS cells were transfected with siControl or siDUB1. After 24 h, the cells were transfected with the Flag-TAZ or Flag vector. After 48 h, total RNA was extracted for gene expression analysis. Each group was tested in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001 for comparisons of target gene expression. E: DUB1 depletion decreased TEAD luciferase activity in MGC803 cells, and this effect was reversed by TAZ overexpression. MGC803 cells were transfected with siControl or siDUB1. After 24 h, the cells were transfected with the Flag-TAZ or Flag vector. After 24 h, the cells were transfected with TEAD luciferase reporter plasmids. The cells were harvested for luciferase activity analysis. F: DUB1 depletion decreased TEAD luciferase activity in AGS cells, and this effect was reversed by TAZ overexpression. AGS cells were transfected with siControl or siDUB1. After 24 h, the cells were transfected with the Flag-TAZ or Flag vector. After 24 h, the cells were transfected with TEAD luciferase reporter plasmids. The cells were harvested for luciferase activity analysis. G: Cell growth inhibition induced by DUB1 silencing was rescued by TAZ overexpression in MGC803 cells. MGC803 cells were transfected with 50 nM DUB1 siRNA or 50 nM control siRNA. After 24 h, the cells were transfected with the Flag-TAZ or Flag vector. After 24 h, a CCK-8 assay was used to determine the cellular metabolic activity at the indicated time points after transfection. Experiments were performed in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001 for comparisons of cell growth. H: Cell growth inhibition induced by DUB1 silencing was rescued by TAZ overexpression in AGS cells. AGS cells were transfected with 50 nM DUB1 siRNA or 50 nM control siRNA. After 24 h, the cells were transfected with the Flag-TAZ or Flag vector. After 24 h, a CCK-8 assay was used to determine the cellular metabolic activity at the indicated time points after transfection. Experiments were performed in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001 for comparisons of cell growth. I-J: DUB1 depletion reduced the number of EdU-positive gastric cancer cells. This effect was further rescued by TAZ overexpression. MGC803 and AGS cells were transfected with siControl or siDUB1. After 24 h, the cells were transfected with the Flag-TAZ or Flag vector. EdU was added to the medium for 2 h of incubation. The absolute cell number was determined to indicate cell proliferation activity. K-L: Cell cycle arrest caused by DUB1 silencing was partially rescued by TAZ overexpression in MGC803 cells. MGC803 cells were transfected with 50 nM DUB1 siRNA or 50 nM control siRNA. After 24 h, the cells were transfected with the Flag-TAZ or Flag vector. The cells were harvested, fixed with 70% ethanol and stained with propidium iodide. The cells were subjected to FACS analysis. Experiments were performed in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001 for comparisons of cell proportions. Representative histograms and cell cycle phase distribution plots are shown in Fig. 4 K and 4L, respectively. M–N: The reduction in the colony formation capacity induced by DUB1 silencing was rescued by TAZ overexpression in MGC803 and AGS cells. Gastric cancer cells were transfected with 50 nM DUB1 siRNA or 50 nM control siRNA. After 24 h, the cells were transfected with the Flag-TAZ or Flag vector. Quantification of colony formation is shown at the indicated time points. The data are presented as the means ± SDs. **P < 0.01, ***P < 0.001 (Student’s t test). O-P: DUB1 depletion decreased the invasive capacity of gastric cancer cells, and this effect was reversed by TAZ overexpression. MGC803 and AGS cells were transfected with 50 nM siControl or siDUB1. After 24 h, the cells were transfected with the Flag-TAZ or Flag vector. After another 24 h, the cancer cells were seeded into the chambers for the Transwell assay. The cell number was determined, and the data are presented as the means ± SDs. **P < 0.01, ***P < 0.001 (Student’s t test). Q-R: DUB1 depletion decreased the migratory capacity of gastric cancer cells, and this effect was reversed by TAZ overexpression. MGC803 and AGS cells were transfected with 50 nM siControl or siDUB1. After 24 h, the cells were transfected with the Flag-TAZ or Flag vector. After another 24 h, the cells were seeded in a 6-well plate until confluent, and a wound was then made by scratching with a sterile tip. Images of the cells were acquired at the indicated time points after scratching