Fig 2: PAR-4 regulates cIAP1 depletion upon DNA damage-induced apoptosis. (a) BT-20 and MDA-MB-468 cells were stimulated with Doxorubicin (10 µM) for 8 h, or Etoposide (100 µM) for 16 h and cell lysates were analysed by immunoblotting using the indicated antibodies. (b) BT-20 cells were treated with Etoposide (100 µM) for the indicated time points and cell lysates were analysed by immunoblotting using specific antibodies for PAR-4, cIAP1, ph p65 Ser536 and GAPDH as control. (c) Par-4 expression in the indicated TNBC cells was silenced as shown in (Fig. 2a) and stimulated with Etoposide (100 µM) for 16 h. Cell lysates were analysed by immunoblotting using antibodies specific for PAR-4, PARP-1, cleaved caspase-8 and GAPDH. (d) BT-20 cells were transiently transfected with myc-tagged cIAP1 WT, cIAP1 H588S mutant for 24 h, and were indicated, cells were treated with Etoposide (100 µM) for 16 h. Cell lysates were analysed by immunoblotting using the indicated antibodies. (a–d) Full-length blots are presented in Supplementary Fig. S6.

Fig 3: Genome-scale gene activation screens identify regulators of necroptosis. a, b Circos plots of genome-scale SAM screens in KBM7 FADD- SAM cells with necroptosis induction by TNFa (a) or SMAC mimetic birinapant (b) for 72 h. For each stimulus, screens were performed at low and high concentrations (TNFa: 10 ng/ml (green) or 100 ng/ml (purple); birinapant: 0.1 µM (blue) or 1 µM (orange)). Screen analysis was performed by identifying differentially enriched sgRNAs using DESeq2 and then aggregating sgRNAs to genes using Gene Set Enrichment Analysis. Identified hits are ranked according to the adjusted p-value of enrichment (–log10(padj). Bubble size corresponds to the average log2 fold-change (aLFC) of enrichment, color indicates the number of significantly enriched sgRNAs. Screens were performed in duplicate except the high concentration of birinapant in simplicate. c, d Cell viability in KBM7 FADD- SAM cells transduced with sgRNA targeting TNIP1, BIRC3 or Renilla luciferase (Ren). Cells were treated as indicated for 72 h and viability was assessed using a luminescence-based readout for ATP (CellTiter Glo). Data represent mean value ± s.d. of two independent experiments performed in triplicates. e KBM7 FADD- SAM cells transduced with the specified sgRNAs were lysed and subjected to immunoblotting with the indicated antibodies. Asterisk (*) indicates non-specific band

Fig 4: (A) LN-229 cells were treated with 50 µM TMZ; 72–120 h later, expression of c-IAP1, c-IAP2, XIAP, Bcl-2, Bcl-xL, and BIRC5/Survivin mRNA was measured by PCR. (B) LN-229 cells were treated with 50 µM TMZ; 120 and 144 h later, expression of Bcl-2, Bcl-xL, c-IAP1, c-IAP2, XIAP, and Survivin protein was measured by immunodetection.

Fig 5: (A) Cell death was measured 120 h upon exposure to different concentrations of TMZ by flow cytometry using PI staining in U87MG cells. (B) Cell cycle distribution was measured 120 h upon exposure to different concentrations of TMZ by flow cytometry using PI staining in U87MG cells. (C) Senescence was measured microscopically by detection of β-Gal positive cells (left panel) or by flow cytometry using C12FDG staining (right panel) 120 h after exposure to 25 and 50 µM TMZ. (D) U87MG cells were treated with 50 µM TMZ. Then, 120 and 140 h later, expression of c-IAP1, c-IAP2, XIAP, Bcl-2, Bcl-xL, and BIRC5/Survivin mRNA was measured by PCR. (E) U87MG cells were treated with 50 µM TMZ for 120 h. Thereafter, the medium was changed, and the cells were either further cultivated for an additional 120 h in the absence or presence of 1, 2.5, or 5 µM BV6 combined with 1, 2.5, or 5 µM venetoclax. After a total of 240 h, cell death was measured by flow cytometry using PI staining in U87MG cells, and Combenefit software was used to perform surface analyses to identify synergy. (A–C) Experiments were performed in triplicate, and differences between treatment and control were statistically analyzed using Student’s t-test (not labeled = not significant, * p < 0.1, ** p < 0.01, *** p < 0.001).