Figure 4

p-ATM and IKKγ interaction and subsequent activation of NFκB or JNK response during genotoxic chemotherapy were largely determined by redox status of resistant cells. (a) Cisplatin-treated cancer cells were flow cytometrically assessed for determining the amount of ROS produced by increasing DCF-DA fluorescence. ROS dependence of apoptosis was validated in A549 cells pre-treated with H₂O₂/NAC. The amount of ROS produced under these conditions was determined flow cytometrically. (b) The fact that IKKγ/p-ATM nuclear co-localization was subject to ROS level modulation was further confirmed by confocal imaging. (c) A549 cells were preincubated with H₂O₂ (4 mM, 1 h)/NAC (40 mM, 12 h) followed by cisplatin treatment, and IKKγ was immunopurified from cell lysates. The immunocomplex was then tested for IKKγ-associated p-ATM by western blotting. In parallel, the levels of p-ATM were determined by western blotting. (d) Co-immunoprecipitation experiments were employed to determine cisplatin-induced IKKγ ubiquitination in H₂O₂/NAC-pre-treated A549 cells. In the same experimental setup, the levels of p-IKKβ, nuclear p65NFκB, p-JNK were also determined by western blotting. (e) Cell viability and ROS production up on treatment of A549 cells with a dose range of cisplatin and H₂O₂-treated were scored by trypan blue dye exclusion assay and flow cytometrically by measuring DCFH-DA fluorescence, respectively. (f) IKKγ precipitates and nuclear lysates of cisplatin-/H₂O₂-treated A549 cells were subjected to western blotting to determine the amount of p-ATM and nuclear p65NFκB, respectively. α-actin and histone H1 were used as internal controls. Values are mean±S.E.M. of five independent experiments in each case. *P<0.01 when compared with respective control sets