Fig. 2. Overproduction of ROS contributes to MNNG-induced parthanatos.

SH-SY5Y cells were treated with MNNG (250 μM) for 4 h with or without 5 mM NAC, 20 nm MitoQ, or 1 μM apocynin pretreatment for 1 h. a CCK8 assay measured cell viability. b FACS analysis of DCFH2-DA fluorescence assessed ROS production. c Quantification of ROS production in all groups. d ELISA measured NADPH activity. e WST-8 assessed Mn-SOD activity. f Western blot analyzed PARP-1 activation and PAR generation. GAPDH expression was used as a loading control. g, h Quantification of PARP-1 (g), and PAR (h) expressions. i Western blot also analyzed AIF amount in both cytoplasm and nuclei. GAPDH or lamin B was used as loading control separately. j Quantification of AIF expression in both cytoplasm and nuclei. Each bar represents the mean ± S.E.M. of three individual experiments. *P < 0.05, **P < 0.01, ***P < 0.001