Figure 3 (See previous page).

ATR-dependent Apaf-1 phosphorylation increases its association with Nup107. (A) Apaf-1 is phosphorylated at serine residue(s) following genotoxic stress. Whole cell extracts derived from A549 cells treated or not with cisplatin were immunoprecipitated with an anti-Apaf-1 antibody and analyzed by immunoblotting with anti-phosphoserine, anti-phosphothreonine or anti-Apaf-1 (as an immunoprecipitation control) antibody. (B) The kinase inhibitor staurosporine prevents Apaf-1 nuclear translocation upon genotoxic stress. MDA-MB436 cells were treated or not with cisplatin (20 μM, 24 h) in the presence or absence of staurosporine (4 nM). After fixation, cells were immunolabeled with anti-Apaf-1 antibody (red). Nuclei were stained with DAPI. Numbers indicate percentage of cells with nuclear Apaf-1 (means ± s.e.m.); n = 3. (C) Staurosporine prevents Apaf-1 phosphorylation upon genotoxic stress. Lysates of cells treated as in (B) were immunoprecipitated with anti-Apaf-1 antibody and analyzed by immunoblotting with anti-phosphoserine or anti-Apaf-1 (as an immunoprecipitation control) antibody. (D) Staurosporine reduces Apaf-1-Nup107 interaction. Lysates of cells treated as in (B) were immunoprecipitated with anti-Apaf-1 antibody and analyzed by immunoblotting with anti-Nup107 antibody. (E) Inhibition of ATR drastically reduces Apaf-1 nuclear translocation upon cisplatin treatment. A549 cells were left untreated or were pretreated for 1 h with DMSO, 10 μM NU-7026, 10 μM KU-55933, 10 μM VE-821, 20 μM U0126, 20 μM SP600125, 50 μM LY294002 or 10 μM BI-D1870. Cells were then treated or not with cisplatin (20 μM, 24 h), fixed and nuclear Apaf-1 assessed by immunofluorescence (means ± s.e.m., n = 3). (F) Inhibition of ATR prevents Apaf-1 phosphorylation upon genotoxic stress. A549 cells pretreated for 1 h with vehicle or 10 μM VE-821 were treated or not with cisplatin (20 μM, 24 h), lysed and the lysates immunoprecipitated and analyzed as in (C). (G) Inhibition of ATR reduces Apaf-1-Nup107 interaction. Lysates of cells treated as in (F) were immunoprecipitated with anti-Apaf-1 antibody and analyzed as in (D). (H) Deregulation of ATR prevents Apaf-1 phosphorylation upon genotoxic stress. A549 cells were transfected with mock or ATR specific siRNAs. After 48 h, cells were treated or not with cisplatin (20 μM, 24 h), lysed and the lysates immunoprecipitated and analyzed as in (C). (I) Deregulation of ATR reduces Apaf-1-Nup107 interaction. A549 cells were transfected with mock or ATR specific siRNAs. After 48 h, cells were treated or not with cisplatin (20 μM, 24 h), lysed and the lysates immunoprecipitated with anti-Apaf-1 antibody and analyzed as in (D). (J) Deregulation of ATR prevents Apaf-1 nuclear translocation upon genotoxic stress. A549 cells were transfected with mock or ATR specific siRNAs. After 48 h, cells were treated or not with cisplatin (20 μM, 24 h). After fixation, cells were immunolabeled with anti-Apaf-1 antibody (red). Nuclei were stained with DAPI. Numbers indicate percentage of cells with nuclear Apaf-1 (means ± s.e.m.); n = 3.