Fig. 1

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