Figure 2
AURKA upregulates Survivin expression and sustains gastric cancer cell survival. (a) shRNA-mediated knockdown of AURKA downregulates Survivin protein levels in AGS and BGC823 cells. Cells were infected with lentiviruses expressing shAURKA or control scrambled RNA targeting GFP. A pool of resistant cells was selected by puromycin and cells were cultured in the presence of 1 μg/ml doxycycline. Forty-eight hours after induction, cells were lysed and subjected to western blotting with the indicated antibodies. (b) AURKA ectopic expression upregulates Survivin protein levels. Cells were transfected with control and AURKA expressing plasmids. After 24 hours, cells lysates were immunoblotted with the indicated antibodies. (c) BGC823 cells were incubated with indicated doses of VX-680 for 24 h before subjected to western blotting with the indicated antibodies. Densitometry was used to quantify the Survivin and GAPDH levels. The relative expression shown (right panel) are means±s.e.m. of the ratios of Survivin to GAPDH. (d) Positive correlation between AURKA and Survivin expression in gastric cancer patients. AURKA and Survivin expression was assessed by immunohistochemistry using gastric cancer tissue samples from 62 patients. Representative staining images of one patient with high AURKA and Survivin and one with low expression are shown. Images (magnification 200 ×). Positive correlation between AURKA and Survivin was observed. (e) BGC823 cells transfected with cDNA encoding wild-type Survivin were treated with or without doxorubicin (1 μg/ml) for 48 h and subjected to Western blot and TUNEL assay. Data represent means±s.e.m. of three independent experiments with significance determined by one-way ANOVA followed by Tukey’s multiple comparison test. (f) BGC823 cells transfected with Survivin siRNA sequences were treated with or without doxorubicin (1 μg/ml) for 48 h and subjected to Western blot and TUNEL assay. Data represent means±s.e.m. of three independent experiments with significance determined by one-way ANOVA followed by Tukey’s multiple comparison test.
