Figure 6. Ku70 knock down enhanced apoptotic cell death after hyperthermia in PC-10.

A. A representative western blot shows the amendment of Ku70 for both siRNAs used. PC-10 cells and H1299 cells were transfected by one siRNA twice. The western result shows significant knock down by Ku70-siRNA-1,-2 comparing with the cont-siRNA-1,-2, respectively. Actin immunoblot was used as a loading control. The experiment was repeated three independent times for reproducibility. PC-10 cells were transfected with Ku70-siRNA-1 or cont-siRNA-1 (200-nM) twice. 24 h after last transfection, equal cell numbers were subcultured for further 24 h, and then treated with hyperthermia for indicated time intervals, and then re-cultured at 37°C for 24 h (B) or 48 h (C) Cells were acquired by FACS analyzer for cell cycle analysis. Results shown are a representative one from, at least, three independent experiments for each time point (24 h and 48 h). D. Ku70 is required for cytostatic arrest by hyperthermia. Histograms show the differential accumulation of cell populations into G2/M phases in cells with or without Ku70, one and two days after 0, 1, 3 and 6 h hyperthermia treatment. Populations in different cell cycle phases, G1, S and G2/M phases, were calculated using computer after hyperthermia treatment. Results shown are average from three independent experiments. E. Significant enhancement of annexin V staining after hyperthermia in Ku70 KD cells compared with control indicating that Ku70 silencing-based cell death by hyperthermia is apoptosis. Each data point represents the mean of three experiments; bars denote SD; * indicates difference from control transfectant at P<0.001.