Figure 6. Mitophagy regulates CSE-induced necroptosis through loss of ΔΨ_m in pulmonary epithelial cells.

(A) Beas-2B cells were incubated for 1 hour with Nex-5 (30 μM), Nec-1 (50 μM), or vehicle (DMSO) and treated with 20% CSE for 15 hours. Cell death was determined by calcein AM/EthD-1 flow cytometry. The x axes show calcein AM staining, and the y axes show EthD-1 staining. Data are representative of 3 experiments. (B) Immunoblot analysis of p-MLKL (Thr357) and total MLKL in lysates obtained from Beas-2B cells treated with 20% CSE at the indicated times. (C) Beas-2B cells were pretreated with control siRNA or PINK1 siRNA for 48 hours prior to treatment with 20% CSE for 8 hours. Cell lysates were immunoblotted for PINK1 and p-MLKL. Total MLKL and β-actin served as the standards. (D) Immunoblot analysis of p-MLKL and total MLKL. Beas-2B cells were incubated for 3 hours with Mdivi-1 (50 μM) or vehicle (DMSO) and treated with 20% CSE for 8 hours. Total MLKL served as the standard. (E) Beas-2B cells were incubated for 1 hour with CsA (10 μM) or vehicle (DMSO) and treated with 20% CSE for 6 hours. Mitochondrial/cytosolic fractions were immunoblotted for PINK1, ubiquitin, RIP1/3, p-MLKL, and MLKL. β-Actin and Tom20 served as the standards. Data represent the mean ± SEM (A). **P < 0.01 by unpaired, 2-tailed Student’s t test (A).