Epstein-Barr Virus Nuclear Antigen 3C Stabilizes Gemin3 to Block p53-mediated Apoptosis
Figure 6
Gemin3 knockdown attenuates EBNA3C-mediated inhibition of p53-induced apoptosis.
(A) Saos-2 (p53−/−) cells were electroporated with different combinations of expression plasmids for HA-p53, EBNA3C-FLAG or vector alone in the presence of small hairpin RNA against Gemin3 (shG3) or control (shCtrl) as indicated. 2×103 transfected cells were cultured in the selection medium (DMEM supplemented with 100 mg/ml G418). After a 2-week selection, cells were fixed on the plates with 4% formaldehyde and stained with 0.1% crystal violet. The area of colonies (pixels) in each dish was calculated by LiCor Odyssey. The number represents the averages of data from two independent experiments. Plasmid expressing RFP was used for normalized transfection efficiency. (B) Western blots showing the protein level of Gemin3 in the lentivirus-mediated Gemin3 or control knockdown cell lines. GAPDH was used as the loading control. (C) Gemin3 knockdown increases apoptosis of EBV negative cells (Ramos and BJAB), EBNA3C positive cells (BJAB7 and BJAB10), and EBV transformed cells (LCL1). Cells were harvested after a 12-h serum starvation and fixed. Levels of cells undergoing apoptosis (sub-G1 phase) in individual PI-stained samples were detected by flow cytometry, and the data were analyzed by FlowJo software. The bar diagram shown at the bottom panel represents the mean of three independent experiments and the results of comparing with each control knockdown sample. (D) Quantitative real-time PCR analysis showed that p53, p21 and Bax genes were upregulated in the Gemin3 knockdown cells. Total RNA was individually isolated from the Gemin3 (shG3) or control (shCtrl) knockdown cells with 12-h serum starvation treatment. A 2 µg total RNA was used to synthesis cDNA. Error bars show standard deviations.