Fig 2: Necrotic NK cell cytotoxicity is triggered by granzyme and mediated by the necroptosis pathway. a Representative phase-contrast and the corresponding GrzmB-FRET reporter images of MCF7 cells in co-culture with primary NK cells with or without 10 nM CMA (vacuolar-type ATPase inhibitor that disrupts lytic granules). Time is indicated in the unit of hours:minutes on the GrzmB-FRET reporter images. The white scale bar is 20 μm. b Distribution of the indicated response phenotypes of MCF7 cells after 6-h co-culture with primary NK cells under the control condition or treatment of 10 nM CMA, 0.9 mM EGTA (calcium chelator that inhibits lytic granule transfer), 40 μM DCI (pan-granzyme inhibitor), and 50 μM Ac-IEPD-CHO (granzyme B inhibitor). c Distribution of the cytotoxic response phenotypes of MCF7 cells upon knockdown of RIP1, RIP3, and MLKL, respectively, and under the treatment of 5 μM GSK’872 (RIP3 inhibitor). The knockdown efficiency is demonstrated by the western blots. Data plotted in b and c were averaged from 3 independent imaging experiments, and the number of cells analyzed for each condition/experiment ranges from 55 to 112. The error bars are standard deviations. Data acquired with NK cells from the same donors are denoted with the same color symbols in each sub-figure. P values were obtained by Student’s t test comparing the treatment condition with control. *P < 0.001. d Left panel: western blot analysis of phospho-MLKL upregulation in MCF7 cells in co-culture with primary NK cells for 3 h and 6 h, respectively. Right panel: representative images of phospho-MLKL immunofluorescence signal in MCF7 cells under control vs. in co-culture with primary NK cells. The white scale bar is 5 μm

Fig 3: 4E-BP Activity Blocks RIPK-Dependent Cytokine Translation(A) TNF-α and CXCL-1 protein levels after indicated stimulations ± 5 IU IFNβ priming overnight in B6, Ifnb−/−, 4E-BP TKO, and RIP1 Ki Ifnb−/− BMDMs.(B and C) TNF-α and CXCL-1 protein levels after indicated stimulations ± treatment with 4EGi-1 (B) or Torin-2 (C) in B6, 4E-BP TKO, and RIP1 Ki Ifnb−/− BMDMs.(D) Cell death as measured by propidium iodide incorporation over 6 h in B6, Ifnb−/−, 4E-BP TKO, and RIP1 Ki BMDMs stimulated with LZ.In all panels, BMDMs were stimulated with LPS, LZ, LZNs, Z, or Ns as indicated. ELISA data are shown as ±SD from three independent experiments compared using two-way ANOVA: n.s. (p > 0.05) and ****p < 0.0001. Kinetic cell death experiments are representative of three or more independent experiments and presented as the mean ± SD of triplicate wells.

Fig 4: BHA acts as a direct RIPK1 kinase inhibitor.A, C Quantitative RIPK1 (AA 1–479) (A) or Ripk3 (AA 1–439) (C) enzymatic activities were measured by ATP consumption using ADP-Glo kinase assays. The compounds were used at indicated concentrations and ‘D’ is short for DMSO. B The ADP-Glo reaction was performed in the absence of active kinase to assess the possible interference of BHA (100 µM) with the luminescence reaction. Results are presented as a percentage relative to the activity of the RIPK1/3 in absence of inhibitors (A, C), or as a percentage relative to the ADP-Glo luminescence reaction (B) and are the mean ± SEM of three independent kinase assays (n = 3). Statistical significance was determined by one-way ANOVA followed by a Tukey post hoc test (A–B) or by a two-tailed paired t test (C). D MEFs were pretreated for 30 min with the indicated compounds (5 µM TPCA-1) before stimulation with 1 µg/ml FLAG-hTNF for the indicated duration. TNFR1 complex I was then FLAG-immunoprecipitated and the IPs were treated with USP21 before analysis by immunoblot, where pRIPK1 refers to autophosphorylation of RIPK1 on S166/T169. The results are representative of at least two independent experiments. E MEFs were pretreated for 30 min with the indicated compounds before stimulation with 20 ng/ml hTNF for the indicated duration. Cell death was measured over time by Sytox Green (SG+) positivity, and the results are presented as mean ± SEM of three independent experiments (n = 3). Statistical analysis on kinetic cell death assays with more than one timepoint is detailed in the Methods section (E). Significance between samples is indicated in the figures as follows: *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant.

Fig 5: Tumor epithelial cell lines null for SERPINB3 undergo lysoptosis-like death under conditions that induce RIP1K-dependent necroptosis.a Percent dead ((# Sytox positive nuclei/# of blue nuclei) × 100) of HT3B3-WT or HT3B3-KO cells 12 h after incubation with DMSO or 10 ng/µL human TNFa + 0.1 µm BV6 SMAC mimetic + 0.5 µm qVD-OPh + 1 µm cyclohexamide (T + S + Q + C). b Percent dead (calculated as in panel a) of HT3B3-WT or HT3B3-KO cells incubated with DMSO, 50 µm nec-1 or 10 µm E64d for 1 h prior to exposure to T + S + Q + C for 8 h. c Immunoblot analysis of RIP kinase 1 (a-RIPK1), phosphorylated RIP kinase 1 (a-pRIPK1), and actin (a-act) in HT29 (control cell line), HT3B3-WT, and HT3B3-KO treated with DMSO (-) or T + S + Q + C (+) for 12 h (black arrowheads denote bands for RIPK1 and pRIPK1 based on molecular mass). d–g Representative confocal images (scale bar = 25 µm) of HT3B3-WT or HT3B3-KO cells incubated with DMSO or T + S + Q + C and stained with Lysotracker Deep Red (white). h Quantification of the lysosomal count per cellular µm2 for the experiment in d–g (=9 fields, compared using a one-way ANOVA with Tukey’s multiple comparisons). i–l To quantitate lysosomal content, HT3B3-WT or HT3B3-KO cells were incubated with 3 kDa Cascade blue, 10 kDa Alexa488, 40 kDa TMR, and 70 kDa Texas red labeled dextrans prior to exposure to either DMSO or T + S + Q + C and imaged using live-cell resonance scanning confocal microscopy (=20 z-planes, =10 fields). The number of lysosomes at each time point (LCt) were normalized to the lysosome count at time zero (LCt0). m AUC for each dextran over time for an experiment in i–l (two-way ANOVA with Tukey’s multiple comparisons test). n–q Representative TEM images (scale bars = 2 µm) of HT3B3-WT or HT3B3-KO cells treated with DMSO or T + S + Q + C. r Quantification of the number of necrotic cells per field (=10 fields) for the experiment in n–q. Unless otherwise noted, a representative of =3 replicates is shown, and the presented means ± SD were compared using a two-tailed t-test (ns not significant, ***P < 0.001, **P < 0.01, *P < 0.05). Original imaging data can be found at https://data.mendeley.com/datasets/gvgnvkw4ct/1. Uncropped immunoblots can be found in Supplementary Fig. 21.