Fig. 2

MLKL and Cx43 were upregulated after TSZ-mediated necroptosis in SH-SY5Y cells. (A, B) Molecular docking analysis of Cx43 and MLKL. (C). Western blots showing the protein expressions of MLKL, Cx43, and VHL in SH-SY5Y cells after transfection with plasmids. (D–F) Histogram showing the quantitative analyses of MLKL, Cx43, and VHL protein levels in SH-SY5Y cells after transfection with plasmids (n ≥ 3 in each group). (G) Western blots showing the protein expressions of Cx43, MLKL, and p-MLKL in SH-SY5Y cells after 8, 10, and 12 h of treatment with TSZ. (H–J) Histogram showing the quantitative analyses of MLKL, pMLKL, and Cx43 protein levels in SH-SY5Y cells after 8, 10, and 12 h of treatment with TSZ (n ≥ 3 in each group). (K) Viability of SH-SY5Y cells after 8, 10, and 12 h of treatment with TSZ (n ≥ 3 in each group). (L) Release of LDH in SH-SY5Y cells after 8, 10, and 12 h of treatment with TSZ (n ≥ 3 in each group). (M) Flow cytometry results: quantitative analysis of necroptotic cells after 8, 10, and 12 h of treatment with TSZ (n ≥ 3 in each group). (N) Representative photographs of flow cytometry analysis of necroptotic cells after 8, 10, and 12 h of treatment with TSZ. Each bar represents the mean ± standard deviation. ^*P < 0.05 vs. control group and ^#P < 0.05 vs. all groups except control group. MLKL, mixed lineage kinase domain-like protein; Cx43, connexin 43; VHL, von Hippel-Lindau; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; LDH, lactate dehydrogenase; TSZ, TNFα + Smac mimetic + ZVAD-FMK; DMSO, dimethyl sulfoxide; PI, propidium iodide