Fig. 2: APIP suppresses cardiac cell death in vitro via the activation of AKT-HIF1α axis.
From: Cardioprotective role of APIP in myocardial infarction through ADORA2B
a, b Suppression of cell death of primary cardiomyocytes in the hypoxic condition. Cardiomyocytes isolated from neonatal WT and APIPTg/Tg mice were exposed to normoxic (Nor) or hypoxic (Hypo) conditions for 36 h (a). The percentage of cell death was determined with the trypan blue exclusion assay (***P < 0.001, one-way ANOVA/Bonferroni, n = 3) (b). c Promoted cell death of Apip-haploinsufficient mouse (Apip+/−) cardiomyocytes under hypoxia. Primary cardiomyocytes were cultured from Apip+/− mice and age-matched WT mice, exposed to hypoxic condition for 24 h, and dead cells were stained with propidium iodide (PI) (***P < 0.001, one-way ANOVA/Bonferroni). d–f Elevation of HIF1α and the phosphorylated AKT by APIP expression under hypoxia. H9c2 cells were transfected with pcDNA3-HA (Mock) or APIP-HA for 24 h, incubated under hypoxia for 6 h, and analyzed by western blotting (d). The signals of HIF1α and p-AKT on the blots were quantified by densitometric analysis (***P < 0.001, *P < 0.05, one-way ANOVA/Bonferroni). N.D. not determined (e, f). g Lack of HIF1α stabilization by Apip knockdown under hypoxia. H9c2 cells were transfected with pSUPER-neo (Mock) or Apip shRNAs (shApip #1 and shApip #2) for 24 h, incubated under hypoxia for 6 h, and analyzed by western blotting. h HIF1α stabilization by APIP depends on AKT and mTOR. H9c2 cells were transfected with pcDNA3-HA or APIP-HA for 24 h, treated with 30 μM LY294002, 10 μM U0126 or 1 μg/ml rapamycin for 2 h, and then incubated under hypoxia for 6 h. i Protection of hypoxic cell death by APIP-AKT requires HIF1α. H9c2 cells were transfected with pcDNA3-HA (Mock) or APIP-HA alone or together with HIF1α-FLAG for 24 h, exposed to hypoxic condition for 48 h in the presence or absence of 30 μM LY294002, and then stained with ethidium homodimer (EtHD) (***P < 0.001, **P < 0.01 one-way ANOVA/Bonferroni, n = 4)
