Figure 3

Depletion of TopBP1 or Claspin enhances radiosensitivity of PC14PE6 cells in vitro . (A) TopBP1, Claspin, and luciferase knock-down cells are regulated by doxycycline treatment through Tet On/Off system. The cells expressing luciferase shRNA were used as a control. To knockdown the target genes, the radio-resistant PC14PE6/shRNA cells were treated with doxycycline (1 μg/ml) for 72 hours and exposed to 10 Gy of IR. The protein levels were evaluated by Western blotting. The phosphorylation of Chk1 S317 and Chk2 Thr68 were induced by exposure of radiation. TopBP1 and Claspin protein levels were significantly down-regulated with the treatment of doxycycline. (B) Cell survival was measured by clonogenic survival assay in TopBP1 or Claspin down-regulated stable PC14PE6 cells with IR treatment as indicated. (C) For overexpression of TopBP1, Claspin, or Chk1, overexpression plasmids were transfected into H460 (a), H23 (b), and U2OS (c) cells. The expression of each protein was shown by Western blot. α-tubulin was used as the loading control. (D) Overexpression of Chk1, TopBP1 or Claspin enhances radioresistance in vitro. Expression vector of each gene was transfected into three kinds of cell lines. The transfected (a) H460 cells, (b) H23 cells, and (c) U2OS cells were irradiated at 2 Gy. Radiosensitivity was evaluated by clonogenic assay. Each experiment in triplicates was repeated three times. Error bars represent the standard deviation. *P-value < 0.05; **P-value < 0.005.