Fig 2: MTAs induce MLKL phosphorylation-dependent necroptosis in L929 fibrosarcoma, both in vitro and in vivo. a Dose-dependent necroptotic cytolysis effect of MTAs on L929 cells. b A panel of 21 MTAs was tested for necroptotic effect on L929 cells. Heat map analysis of cell death index was calculated based on ATP levels. c Fluorescent microscopy of SYTOX Green-labeled necroptotic L929 cells after NCZ treatment for 24 h. Plasma membrane breakdown was traced by SYTOX Green staining. Scale bar, 400 µm. d Immunoblotting analysis of MLKL phosphorylation by Triton X-114 fractionation in whole cell lysates of NCZ-treated or PTX-treated L929 cells. T, 20 ng/ml recombinant/soluble TNF treatment. Aq, aqueous fraction; Det, detergent fraction. e Effect of Rip3 knockout on MTA-induced necroptosis in L929 cells. f Effect of RIP3 kinase activity on MTA-induced necroptosis in L929 cells. Wild-type or mutants of RIP3 were stably expressed in Rip3 KO L929 cells by pHAGE infection. WT, wild-type RIP3; K51A, kinase dead form of RIP3; S232A, auto-phosphorylation site mutant of RIP3. RIP3 re-expression was detected by immunoblotting. g In vivo response of mouse allograft of L929 cells to VCR. Athymic nude mice bearing ~300 mm3 L929-fibrosarcoma were treated with vehicle or with 5 mg/kg Nec-1s and/or 5 mg/kg VCR. Upper: tumor growth was measured and calculated. Lower: representative image of L929 cells allografts on day 6. Vehicle, n = 5; VCR, n = 7; VCR + Nec-1s, n = 5. Scale bar, 1 cm. Graph shows mean ± SEM, p values were determined by the two-way ANOVA test; NS not significant; *p < 0.05; **p < 0.01; ***p < 0.001. D, DMSO; NCZ, nocodazole; VCR, vincristine; PTX, paclitaxel; DTX, docetaxel. Cell viability was determined by measuring ATP levels. The data are represented as mean ± SEM of duplicate wells (a, b, e, and f). Results are reported from one representative experiment. Experiments were repeated independently for four (a, c), three (d–g), or two (b) times

Fig 3: SB216763 reduces the levels of necroptosis-related proteins in astrocytes. Astrocytes were exposed to OGD for 6 h and reoxygenation for 24 h. Astrocytes were treated with or without SB216763 (5 µM) during OGD and reoxygenation. (A–C) Representative images from Western blotting (WB) analysis of the levels of RIP1K, p-RIP1K, RIP3K, p-RIP3K, MLKL, and p-MLKL. Columns present data from the quantitative analysis of RIP1K, p-RIP1K, RIP3K, p-RIP3K, MLKL, and p-MLKL immunoblots, respectively. ß-Actin protein was used as a loading control. Mean ± SD, n = 3. **P < 0.01, *P < 0.05 vs. non-OGD-Re24 h group; ##P < 0.01, #P < 0.05 vs. OGD6 h-Re24 h group.

Fig 4: Hepatic vFlip expression triggers regulated necrosis in hepatocytes.a–e Representative data derived from neonatal liver tissue of vFlipAlbCre+ (n = 5) and control littermates (n = 6). Experiments were repeated three times with similar results. a Representative stainings of H&E, TUNEL (red), cl. CASP3 (green), MLKL (red), GSDMD (red) counterstained with Hoechst (blue, nuclei) in consecutive liver cross-sections. Scale bar: 100 µm. b Relative mRNA expression of Mlkl in the liver. c Detection of hepatic MLKL and GSDMD protein level by Western Blot. GAPDH was used as loading control. d Representative images via z-stack confocal microscopy from mock, Etanercept (TNF inhibitor) and Necrostatin-1 (RIPK1 inhibitor) treated liver organoids derived from embryonic vFlipAlbCre+ and control mice. Scale bar: 100 µm. e, f Relative mRNA expression of genes involved in e pyroptosis and f lipid peroxidation in the liver of indicated mice. g Detection of GPX4 protein level by Western Blot. GAPDH was used as loading control. Gene expression levels are shown relative to Hprt. Error bars indicate ± SD, ***P < 0.001, ****P < 0.0001 by unpaired two-tailed t-test.

Fig 5: ADAM17 hypomorphic MEF are protected from TNF-induced necroptosis(A) MEF from WT and ADAM17ex/ex mice were left untreated or preincubated for 30 min with 20 µM zVAD-fmk in the absence or presence of 1 µg/ml CHX, followed by stimulation with 100 ng/ml TNF. After 16 h, loss of membrane integrity was determined as a marker for cell death by flow cytometric analysis of PI-positive cells. Each measurement represents the mean of three parallel determinations, error bars indicate the corresponding SD. (B) in parallel, cell lysates were analyzed by Western blot for presence of pMLKL, MLKL, pRIPK3, RIPK3, cleaved caspase-3 and pBcl-2. Detection of actin served as a loading control. (C) WT MEF were left untreated or preincubated for 30 min with 3 µM GW280264X (GW), 3 µM GI254023X (GI), 10 µM marimastat (M) or 50 µM TAPI-1 (T) in combination with zVAD-fmk, CHX and subsequent addition of TNF as in A before cell death was analyzed. Each measurement represents the mean of three parallel determinations, error bars indicate the corresponding SD. ns, not significant, *, p<0.05, **, p<0.01, ***, p<0.001 (two-tailed unpaired Student’s t-test).