Augmented LUBAC activity overcomes cell death induced by DNA damage thereby accelerating accumulation of somatic mutations. (A) Transcript levels of Aicda, normalized to Actb (left panel), and percentages of germinal center B cells (right panel) in mesenteric lymph nodes from 10-week-old mice; n = 3 per genotype. Data represent means ± SD. (B) HOIP-overexpressing HBL1 cells were established, and immunoblot analyses were performed using lysates from wild-type (WT), mock-transfected, or HOIP-overexpressing HBL1 cells. (C) Live cells were analyzed by fluorescence-activated cell sorting using TO-PRO-3 staining. HBL1 cells were treated with or without 10 μg/mL cisplatin for 0 to 24 hours. (D) Percentage of live cells (±SD); n = 6 per group in 3 independent experiments. (E) Immunoblot analyses were performed using lysates from wild-type, HOIP-knockout (KO), or HOIP-overexpressing Jurkat cells. (F) Live cells were analyzed by fluorescence-activated cell sorting using FSC and TO-PRO-3 staining. Jurkat cells were treated with or without 0.5 μg/mL cisplatin for 0 to 72 hours. (G) Percentage of live cells (±SD) in 3 independent experiments. (H-J) Jurkat cells were treated with 3 μg/mL cisplatin for the indicated periods, followed by immunoblotting (H-I) or quantitative reverse-transcription polymerase chain reaction, normalized against Actb messenger RNA (mRNA) (J). (K) Jurkat cells were treated with 5 μg/mL cisplatin for the indicated periods. Whole-cell lysates were analyzed by anti-NEMO immunoprecipitation (IP), followed by immunoblotting using antibodies against linear polyubiquitin (Ub) and NEMO. (L) Correlation of expression of HOIP (RNF31) and negative regulation of intrinsic apoptotic signaling signature (left) and NF-κB signaling signature (right). *P < .05, **P < .01, and ***P < .001, 1-way analysis of variance (ANOVA) with Turkey’s post hoc test (A), 2-way ANOVA with Bonferroni post hoc test (D, G, and J), or Pearson’s correlation (L). See also supplemental Figures 4 and 5 and supplemental Tables 1 to 4.