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. 2020 Feb 19;11(2):134. doi: 10.1038/s41419-020-2328-0

Fig. 6. Comparison of the inhibitory effect of Nec-1s, GSK’872 and NSA on necroptosis in vitro.

Fig. 6

a–c HaCaT cells were treated with TNF-α (100 ng/mL) and Smac (100 nM) for 8 h to induce apoptosis (hereafter abbreviated as TS). Necroptosis of HaCaT cells was induced by TNF-α (100 ng/mL), Smac (100 nM), and z-VAD-fmk (20 μM) for 8 h (hereafter abbreviated as TSZ). The necroptosis cells were treated separately with the RIPK1 inhibitor Nec-1s (10 μM), the RIPK3-inhibitor GSK’872 (10 μM), and the MLKL-inhibitor NSA (5 μM). a Representative transmission electron microscopy (TEM) pictures of HaCaT cells treated as above. Representative images are shown. Scale bars represent 1 μm. b Protein levels of RIPK1, RIPK3, MLKL, pMLKL (S358) and HMGB1 were analyzed by western blotting. GAPDH was used as a loading control. c The mRNA level of IL-1β, IL-6, IL-8, and CXCL1 was analyzed by real-time quantitative PCR. d MLKL oligomerized in membrane faction upon necrosis induction. The cells were harvested and then separated into cytoplasmic and membrane proteins. Protein levels of MLKL and pMLKL (S358) were analyzed by western blotting. GAPDH was used as cytoplasm protein control. N-cadherin was used as membrane protein control. Results shown are representative data of three independent experiments. Error bars represent mean ± standard deviation (SD). ns p > 0.05, *p < 0.05, **p < 0.01, and ***p < 0.001 when compared.