Fig 1: RelA mediates protection from necroptosis through TNF-inducible A20 A, BFractional survival quantified by crystal violet assay after treatment with TNF and (A) ROS inhibitor (ROSi), (B) JNK inhibitor (JNKi), and/or ZVAD (mean of three independent experiments ± standard deviation; two-tailed Student's t-test *P < 0.05, **P < 0.01, ***P < 0.001, or no statistically significant difference, n.s., P > 0.05).CTNF-induced death rates in L929 wild-type cells with and without ZVAD pre-treatment obtained via live-cell microscopy (mean of three independent experiments ± standard deviation).DTNF-induced mRNA expression measured via qRT–PCR (mean of three independent experiments ± standard deviation; two-tailed Student's t-test revealed no statistically significant results, P > 0.05).EImmunoblot for FLIP-L (full) and its cleavage product (p43; representative data of three independent experiments).FRelative mRNA expression of A20 or cIAP2 measured via qRT–PCR, and normalized RelA activity measured via EMSA in wt cells treated with TNF (mean of three independent experiments ± standard deviation).GImmunoblot for A20 protein (representative data of three independent experiments).HWhole cell lysates depleted of RIPK3-containing protein complexes using anti-RIPK3 served as input for RIPK1 co-immunoprecipitation (IP) and subsequent immunoblot analysis. Flow through, FT. Isotype control, IgG rb.IRepresentative microscopy images of smFISH experiments for the NF?B-responsive target genes A20 (red spots) and I?Ba (green spots) in wt cells treated with TNF (contrast set to same limits for every time point).JHistogram of volume normalized mRNA copy numbers measured by smFISH (two independent experiments; first independent experiment shown in Fig 2D).K, L(K) Immunoblot for cIAP2 using commercially available Pan-cIAP antibody and cIAP1-knockout cell line as control. As cIAP2 protein is below detection limit, CRISPR/Cas9-mediated knockout was validated (L) via high-resolution melt (HRM) analysis.MTNF-induced death rates obtained via live-cell microscopy (mean of three independent experiments ± standard deviation). Source data are available online for this figure.