Figure 5

Reduced ATM-dependent activation of NF-κB augmented radiation-induced apoptosis by G α s. (A) Effect of NF-κB inhibitors on radiation-induced cleavage of caspase 3 and PARP in H1299 cells. (B) Effect of constitutively active IKKα (IKKαSE) and IKKβ (IKKβSE) on on radiation;-induced cleavage of caspase 3 and PARP in H1299 cells. (C, D) Effect of Gαs on the activation of NF-κB in H1299 cells. The graph was made from the western blot band densities (empty bar: nuclear p50, filled bar: nuclear p65). (E) Effect of Gαs on NF-κB-dependent luciferase activity in H1299 cells. The empty bar represents vector control and the filled bar GαsQL transfected cells. (F) Effect of ATM activity on NF-κB-dependent luciferase activity in H1299 cells. (G) Effect of Gαs on NF-κB-dependent luciferase activity in A549 cells (H) Effect of Gαs on cytosolic pATM. Asterisks (*) on the histograms indicate a statistically significant difference from the vector-transfected control cells; the double asterisks (**) represent a statistically significant difference from the irradiated control cells; the triple asterisks (***) represent a statistically significant difference from the GαsQL-transfected control cells (p < 0.05, Mann–Whitney U test). H1299 cells were pretreated with KU55933 (10 μM), or PDTC (5 μM), BAY 11–7082 (10 μM), or IKK inhibitor VII (IKKi VII, 500 μM) for 30 min or chloroquine (20 μg/ml) for 4 h with/without transfection of GαsQL or vector. Then, the cells were irradiated with γ-rays (10 Gy) and incubated for 24 h. NF-κB-dependent luciferase activity was assessed using the Dual-Luciferase Reporter Assay System. The cells were also lysed and fractionated into nucleus and cytosol fraction before western blot analysis. PARP was the markers for nucleus fraction.