NPM1 is required for PIDDosome signaling. (A) HeLa.C2 Pro-BiFC cells were transfected with the indicated siRNAs in the presence of qVD-OPH (20 µM). 48 h later, cells were treated with or without camptothecin (100 µM). The percentage of cells that were Venus⁺ in the nucleolus, nucleus, or cytoplasm was determined at 24 h from at least 50 microscopy images per well. Results are the mean of three independent experiments ± SD. *, P < 0.05. (B) Representative confocal images show caspase-2 BiFC (yellow) and mCherry expression (red) from cells treated as in A. Bars, 10 µm. (C) HeLa.C2 Pro-BiFC cells transfected with the indicated siRNAs in the presence of qVD-OPH (20 µM) for 48 h were treated with or without IR (50 Gy) ± Gö6976 (1 µM). The percentage of cells that were Venus⁺ in the nucleolus, nucleus, or cytoplasm was determined at 24 h from at least 50 microscopy images per well. Results are the mean of four independent experiments ± SD. ***, P < 0.0005. (D) Tp53^−/− MEFs of indicated Npm1 genotypes were treated with or without Gö6976 (1 µM) ± IR (10 Gy) and harvested 24 h later. Lysates were analyzed by Western blot. (E) Tp53^−/− MEFs of indicated Npm1 genotypes were treated with or without camptothecin (100 or 150 µM) and harvested 24 h later. Lysates were analyzed by Western blot. (F) PC3 cells transfected with the indicated siRNAs were treated with or without Gö6976 (1 µM) ± IR (10 Gy) and harvested 24 h later. Lysates were analyzed by Western blot. (G) OCI-AML2 and OCI-AML3 cells, of indicated NPM1 genotypes, were treated with or without Gö6976 (1 µM) ± IR (10 Gy) and harvested 24 h later. Lysates were analyzed by Western blot.