Figure 4.

Role of ATM and AMPK in the signalling and antiproliferative effects of MET and IR. (A–C) Ataxia telengiectasia-mutated regulates AMPK in response to MET and IR. A549 cells were either transfected with ATM-specific siRNA or control vector and incubated for 72 h (B) or incubated with the ATM-specific inhibitor KU60019 or vehicle for a period of 24 h (C), before treatment with 5 μℳ MET for 48 h (A–C) and/or IR of 0, 2 or 8 Gy 24 h (C) after initiation of MET treatment. After treatments, cells were washed, lysed and probed with indicated antibodies. Representative immunoblots of three independent experiments are shown. AMP-activated kinase mediates the signalling and antiproliferative effects of MET and IR. A549 cells were pretreated with siRNAs against (D) AMPKα1 and AMPKα2 catalytic subunits or control vector for a period of 72 h before treatment with 5 μℳ MET for a 48-h period and/or IR dose of 0, 2 or 8 Gy for a 24-h period. After treatment, cells were washed, lysed and probed with indicated antibodies. Representative immunoblots of three independent experiments are shown. (E) A549 cells were pretreated with control vector (vehicle) or siRNA sequences against AMPKα1 and AMPKα2 catalytic subunits for a period of 72 h before a 48-h treatment with MET (0 μℳ–1 mℳ) and a 24-h treatment with 0, 2 or 8 Gy dose of IR. Proliferation results (mean±s.e.) of three independent experiments (six replicates per condition in each experiment) are shown. (F) WT and AMPKα1/2^−/−-MEFs were treated with MET (0 μℳ–100 μℳ) for a period of 48 h. After treatment, cells were washed, lysed and probed with indicated antibodies. Representative immunoblots of three independent experiments are shown. (G) WT and AMPKα1/2^−/−-MEFs were treated with MET (0 μℳ–5 mℳ) for a period of 48 h and/or the indicated doses of IR for 24 h. Proliferation results (mean±s.e.) of three independent experiments are shown.