Figure 5

Effect of Akt isoforms and DNA-PKcs on post-irradiation cell survival of K-RAS-mutated A549 cells. (a) Forty-eight hours after the transfections with the indicated siRNAs, the cells were plated in six-well plates, the colonies were stained after about 10 days and the plating efficiencies were calculated by dividing the number of colonies formed to the number of cells seeded. The data presented are the mean plating efficiencies (PE)±S.E.M. of 12 replicates from two independent experiments. (b) Transfected cells with indicated siRNA were plated and X-ray irradiated 24 h later and then incubated for 10 days. Thereafter, the colonies were stained, and the survival fractions (SF) were calculated as described in the Materials and Methods section. The data presented are the mean survival fraction±S.E.M. of 12 replicates from two independent experiments. (c) Confluent A549 cells were treated with the vehicle (DMSO) or the DNA-PKcs inhibitor NU7026 at indicated concentrations for 1 h and then irradiated with 4 Gy. Protein samples were isolated 30 min after irradiation, and levels of P-DNA-PKcs (Ser-2056) and P-DNA-PKcs (Thr-2609) were determined by immunoblotting. The blots were then stripped and incubated with the DNA-PKcs antibody. (d) A549 cells were plated in six-well plates and 24 h later were treated with the vehicle (DMSO) or the indicated concentrations of the DNA-PKcs inhibitor NU7026 for 1 h. The cultures were then irradiated and incubated for 10 days. Thereafter, the colonies were stained, and the clonogenic fractions were calculated as described in Materials and Methods section. The data presented are the mean survival fraction±S.E.M. of six replicates from the parallel experiments. The asterisks indicate a statistically significant inhibition of plating efficiency (a) and radiosensitization after knockdown of Akt1 or Akt3 (b) (*P<0.05; **P<0.01; ***P<0.001).