Fig. 5.

ARC prevented mPTP opening and necrotic cell death by inhibiting the activity of CypD. (A) Colocalization of ARC with CypD in cardiomyocytes was detected by immunofluorescence staining. ARC, green; CypD, red; nuclei, blue; scale bar, 10 µm. (B) Interaction of ARC with CypD was detected by immunoprecipitation in 293 T cells with ectopic expression of HA-ARC and Myc-CypD. (C) Interaction of endogenous ARC with CypD in cardiomyocytes was detected by immunoprecipitation. (D) Overexpression of ARC inhibited necrosis in cardiomyocytes, which was abolished by simultaneous overexpression of CypD in cardiomyocytes exposed to 500 μM H₂O₂ for 12 h. Necrosis was detected by PI assay. Error bars represent SEM; *P < 0.05 vs control. Knockdown of ARC sensitized cardiomyocytes to undergo necrosis which was inhibited by simultaneous knockdown of CypD in cardiomyocytes exposed to 100 μM H₂O₂ for 12 h. Necrosis was detected by PI assay (E) and indicated by LDH release assay (F). Error bars represent SEM; *P < 0.05 vs control. Forced expression of CypD sensitized cardiomyocytes to undergo necrosis which was abolished by simultaneous overexpression of ARC. Necrosis was detected by PI assay (G) and indicated by LDH release assay (H). Error bars represent SEM; *P < 0.05 vs control.