Fig. 4

NPRL2 depletion induces DNA damage and cell cycle arrest. A Left, NPRL2 depletion led to accumulation of a DNA damage marker, cell cycle inhibitors, and an apoptosis marker. Ras-AI-T2 cells were transduced with control or NPRL2-targeting shRNA-containing virions (20 μL virion-containing medium/10⁴ cells) for 48, 72 and 96 h. Then, cell lysates were used for immunoblot analyses. Right, paramycin might decrease NPRL2 depletion-induced cell cycle arrest and apoptosis. Ras-AI-T2 cells were transduced as described above and treated with 20 nM rapamycin (Rap.). The cells were then subjected to immunoblotting analyses. B NPRL2 depletion increased the percentages of cells in G1 and G2/M phases. Upper, After transduction for 48, 72 or 96 h, Ras-AI-T2 cells were collected and fixed for cell cycle analysis (lower). Alternatively, the transduced cells were treated with or without 20 nM rapamycin (Rap.) for 48 or 96 h and then collected for analysis of cell cycle distribution (lower). Results are the mean ± SD determined from three experiments. *p < 0.05 by Student’s t-test