Fig. 3

DUB1 depletion inhibits gastric cancer progression in vivo and in vitro. A-B: Depletion of DUB1 inhibited the proliferation of gastric cancer cells. MGC803 and AGS cells were transfected with siControl or siDUB1. Two different siRNAs were used. After 24 h, a WST-1 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. C-D: DUB1 depletion reduced the number of EdU-positive gastric cancer cells. MGC803 and AGS cells were transfected with siControl or siDUB1. After 24 h, EdU was added to the medium for 2 h of incubation. The absolute cell number was determined to indicate cell proliferation activity. E–F: Cell cycle analysis was performed to assess the effect of DUB1 silencing on MGC803 cells. MGC803 cells were transfected with 50 nM DUB1 siRNA or 50 nM control siRNA. After 24 h, 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. 3E and 3F, respectively. G-H: DUB1 promoted the migration of MGC803 and AGS gastric cancer cells. MGC803 and AGS cells were transfected with siControl or siDUB1. After 24 h, Transwell assays were used to evaluate the migratory capacity. The cell number was determined, and the data are presented as the means ± SDs. **P < 0.01, ***P < 0.001 (Student’s t test). I-J: Wound healing assay of MGC803 and AGS cells with DUB1 depletion or siControl transfection. Quantification of wound closure at the indicated time points. The data are presented as the means ± SDs. **P < 0.01, ***P < 0.001 (Student’s t test). K-M: DUB1 depletion inhibited gastric tumor growth in vivo. MGC803 cells were stably transduced with lentiviral vectors expressing scrambled shRNA or DUB1 shRNA. These MGC803 cells (2 × 10⁶) were injected into the right dorsal flanks of 4-week-old female BALB/c nude mice. Tumor formation in the nude mice was monitored over a 4-week period. The tumor volume was calculated with the following equation: tumor volume = 0.5 × length × width². Mice were sacrificed five weeks after tumor cell injection. Tumor growth curves, weights and photographs are shown in Panels K, L and M, respectively