Fig. 7: Atg13 interacts with FADD to activate caspase-8 under hypoxia.

a H9c2 cells were infected with adenoviral constructs expressing Atg13 (Ad-Atg13) or β-gal (Ad-β-gal) and then treated with 10 μM histamine accompanied by DMSO or 20 μM Z-VAD-fmk for 24 h. Quantitative analysis of apoptosis detected by TUNEL assay is shown. b H9c2 cells were treated with 10 μM histamine under hypoxia for 24 h. The activities of caspases as indicated were determined by the corresponding kits. c H9c2 cells were treated with 10 μM histamine along with 1 μM pyrilamine under hypoxia for 24 h. The activation of caspase-8 and caspase-3 was determined by immunoblot. d H9c2 cells were infected with adenoviral Atg13 (Ad-Atg13) or were transfected with miR-206/216b antagomirs (miR-206/216b Anta) for 8 h prior to 10 μM histamine treatment under hypoxia for 12 h. The activation of caspase-8 and caspase-3 was determined by immunoblot analysis. e H9c2 cells were infected with adenoviral Atg13 (Ad-Atg13) or β-gal (Ad-β-gal) and then treated with 10 μM histamine accompanied by DMSO as control, 20 μM Z-VAD-fmk or 10 μM Z-IETD-fmk for 24 h. Quantitative analysis of apoptosis detected by TUNEL assay is shown. f H9c2 cells were treated with 10 μM histamine and 2 mM 3-methyladenine (3-MA) under hypoxia for 24 h. The activation of caspase-8 and the formation of LC3II were determined by immunoblot. g H9c2 cells were treated with 110 nM Atg13-siRNA (Atg13-Si) or its negative controls (Scramble) for 8 h prior to 10 μM histamine treatment for 24 h. The activation of caspase-8 and the level of Atg13 were determined by immunoblot. h H9c2 cells were infected with adenoviral Atg13 (Ad-Atg13) or β-gal (Ad-β-gal) and then treated with 10 μM histamine under hypoxia for 24 h. Immunoprecipitation was performed using an Atg13 antibody. The anti-IgG served as a control. The levels of Atg13, FADD, pro-caspase, and cleaved caspase-3 were analyzed by immunoblot. i H9c2 cells were infected with adenoviral Atg13 (Ad-Atg13) for 8 h prior to 2 mM 3-methyladenine (3-MA) treatment under hypoxia for 24 h. Immunoprecipitation was performed using an Atg13 antibody. The anti-IgG served as a control. The levels of Atg13, FADD, and pro-caspase were analyzed by immunoblot.  j H9c2 cells were infected with adenoviral Atg13 (Ad-Atg13) accompanied by 90 nM FADD siRNA (FADD-Si) or its negative controls. Then cells were treated with 10 μM histamine under hypoxia for 24 h. Immunoprecipitation was performed using an Atg13 antibody. The anti-IgG served as a control. The levels of Atg13, FADD and pro-caspase were analyzed by immunoblot. k H9c2 cells were transfected with 90 nM FADD-Si or its negative controls. Then cells were treated with 10 μM histamine under hypoxia for 24 h. The activation of caspase-8 and the level of FADD were determined by immunoblot. *P < 0.05; **P < 0.01