Fig. 5

Interaction of MLKL Ser454 with Cx43 can trigger the opening of Cx43 hemichannels, leading to increased intracellular Ca²⁺, and cell necroptosis. A, B) Western blots to detect MLKL and pMLKL polymers in SH-SY5Y cells post TSZ treatment, Cx43, MLKL(WT), and MLKL(S454A) transfection. C (a-h) Dye uptake assay to detect Cx43 hemichannels in SH-SY5Y cells post TSZ treatment, Cx43, MLKL(WT), and MLKL(S454A) transfection. C (i-p) Fluo-4 AM to detect cytoplasmic Ca²⁺ concentration in SH-SY5Y cells post TSZ treatment, Cx43, MLKL(WT), and MLKL(S454A) transfection (scale: 50 μm). D) The histogram presents the quantitative analyses of EB fluorescence intensity (n = 6 in each group). E) The histogram presents the quantitative analyses of Fluo-4 AM fluorescence intensity (n = 6 in each group). MLKL, mixed lineage kinase domain-like protein; Cx43, connexin 43; WT, wild type; MT, mutant; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; TSZ, TNFα + Smac mimetic + ZVAD-FMK; EB, ethidium bromide