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

Fig. 2. Inhibition of RIPK1 attenuated RIPK1/RIPK3- mediated necroptosis in vitro.

Fig. 2

a–h HaCaT cells were treated with TNF-α (100 ng/mL), Smac (100 nM), and z-VAD-fmk (20 μM) for 8 h (hereafter abbreviated as TSZ) to induce necroptosis and then stimulated with Nec-1s (10 μM). a Cell viability was measured by the Cell Counting Kit-8 (CCK-8) assay. b LDH release was measured by the cytotoxicity LDH assay kit. c Protein levels of RIPK1, RIPK3, pRIPK3(S227), MLKL and pMLKL (S358) were quantified by western blotting. GAPDH served as the loading control. d mRNA level of RIPK1, RIPK3, and MLKL was determined by real-time quantitative PCR in the three groups. e–h Real-time quantitative PCR was performed to analyze the mRNA level of IL-1β (e), IL-6 (f), CXCL1 (g), and IL-8 (h). i, j Knockdown of RIPK1 by shRNA in HaCaT cells was confirmed by western blotting (i) and real-time quantitative PCR (j). k, l After RIPK1-knockdown in HaCAT cells, they were treated with TSZ. Cell viability was measured by the Cell Counting Kit-8 (CCK-8) assay. LDH release was measured by the cytotoxicity LDH assay kit. Error bars represent mean ± standard deviation (SD). ns p > 0.05, *p < 0.05, **p < 0.01, and ***p < 0.001 when compared. All the assays were repeated three times and the results were consistent.