Figure 1

Gαs inhibited ATM activation induced by γ−ray irradiation in lung cancer cells. (A) Effect of Gαs on the phosphorylation of ATM following γ-ray irradiation in H1299 lung cancer cells. (B) Effect of Gαs on the proteins involved in DNA damage responses following γ-ray irradiation in H1299 cells. L represents the long forms of Gαs, and S represents the short forms of Gαs. (C) Densitometric analysis of the phosphorylation of ATM and H2AX. The histograms represent the means and standard errors of at least three independent experiments (empty bar: p-ATM, filled bar: ATM, diagonal bar: γ-H2AX, and hatched bar: H2AX), and an asterisk (*) indicates a statistically significant difference from the vector-transfected control cells (p < 0.05, Mann–Whitney U test). (D) Effect of Gαs on the phosphorylation of ATM following γ-ray irradiation in A549 lung cancer cells. (E) Subcellular fractionation analysis of ATM phosphorylation. (F) Confocal microscopic analysis of ATM phosphorylation. Lung cancer cells (H1299 and A549 cells) were transfected with EE-tagged GαsQL or a pcDNA3 vector (V), incubated for 24 h, and irradiated with γ-rays (5 Gy). After incubation for 30 min or for the indicated times, the expression and phosphorylation of the proteins involved in DNA damage responses were analyzed by western blotting. Each lane represents cells that were separately transfected, and β-actin was used as a loading control. Thirty minutes after irradiation, the cells were lysed and fractionated for western blotting. COX-1 and PARP were used as markers for cytosolic and nuclear fractions, respectively. One hour after irradiation, phosphorylated ATM was assessed by staining with p-ATM-FITC (green) and DAPI (blue), and the samples were then analyzed by confocal microscopy.