Figure 5

Degradation of PIASγ at high ROS during genotoxic stress interrupts the ATM/IKKγ interaction with subsequent activation of NFκB in resistant cells. ROS dependency of (a) PIASγ/IKKγ interaction and (b) IKKγ sumoylation were analyzed in A549 cells treated with H₂O₂ (4 mM, 1 h)/NAC (40 mM, 12 h) prior to cisplatin, by co-immunoprecipitation and Western blotting. (c) PIASγ immunoprecipitates from H₂O₂/NAC pre-incubated cisplatin-treated A549 cells were probed with anti-DNP and anti-ubiquitin antibodies to determine the levels of PIASγ oxidation and degradation (upper left panel). Total PIASγ level was determined in MG132 pre-incubated and cisplatin-/H₂O₂-treated A549 cells (lower panel). Flow cytometric analyses of the same experimental set further determined percent apoptosis by Annexin-V-/7-AAD-positivity (right panel). (d) Co-immunoprecipitation to assess the IKKγ sumoylation, IKKγ-pATM interaction and western blotting analysis to determine p65NFκB nuclear translocation in untransfected/PIASγ-siRNA-/PIASγ-cDNA-transfected cells in presence and absence of cisplatin. (e) Flow cytometric analyses of the same experimental set for assessing percent apoptosis by Annexin-V-/7-AAD-positivity (upper panel). The effectiveness of PIASγ genetic modifications was validated by both western blot analysis and RT-PCR (lower panel). (f) Percent apoptosis was scored in A549 cells transfected with the catalytically inactive PIASγcDNA (PIASγCA-cDNA)/WT-A549 cells using Annexin-V-/7-AAD flow cytometric assay (upper panel). The effectiveness of PIASγCA transfections was validated by western blot analysis (inset). The western blotting technique was employed to examine p65NFκB nuclear translocation in un-transfected/PIASγCA-cDNA-transfected cells in presence and absence of cisplatin (lower panel). (g) Percent viability of different cancer cells treated with dose ranges of cisplatin and doxorubicin was scored by using trypan blue dye exclusion assay. (h) Untreated-/Cisplatin (5 μM)-treated cancer cells were assessed flow cytometrically by monitoring the increase in DCF-DA fluorescence for amount of ROS produced. (i) H₂O₂-pre-treated-DS-MCF-7 and NAC-pre-treated-MCF-7 cells were estimated flow cytometrically by measuring DCFH-DA fluorescence (left panel) and Annexin-V-/7-AAD-positivity (right panel). (j) Western blot analysis for the levels of p-ATM, nuclear p65 and p-JNK in un-/cisplatin (5 μM) treated MCF-7 and DS-MCF-7 cells. 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 un-transfected/normal sets