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. 2019 Jul 1;10(7):511. doi: 10.1038/s41419-019-1746-3

Fig. 4. The CREB-HIF1α signaling triggered by ADORA2B depends on APIP.

Fig. 4

a Essential role of APIP in ADORA2B-mediated cytoprotection against hypoxia. H9c2 cells were transfected with pSUPER-neo (Mock) or Apip shRNAs (shApip #1 or shApip #2) for 24 h and incubated with serum-free medium for 24 h. Cells were then treated with 10 μM NECA or 10 μM BAY 60-6583 for 10 min and incubated under hypoxic condition for 24 h (***P < 0.001, N.S., not significant (P > 0.05), one-way ANOVA/Bonferroni). b Role of APIP in ADORA2B-mediated CREB activation under hypoxia. H9c2 cells were transfected with pSUPER-neo (Mock) or Apip shRNAs (shApip #1 or shApip #2) for 24 h and incubated with serum-free medium for 24 h. Cells were then left untreated or treated with 10 μM NECA for 10 min and incubated under the hypoxic condition for 2 h. Cell lysates were analyzed by western blotting and the signals of p-CREB were quantified by densitometric analysis (n = 4, **P < 0.01, *P < 0.05, one-tailed t test). c, d APIP regulates HIF1α stabilization and cell death through CREB under hypoxia. H9c2 cells were transfected with dominant negative CREB (DN-CREB) and APIP-HA for 24 h and incubated under the hypoxic condition for 3 h (c) and 24 h (d). Cell lysates were analyzed by western blotting (c) and cell death rates were determined (***P < 0.001, one-way ANOVA/Bonferroni, n = 4) (d). e APIP protects cells against hypoxia through PKA. H9c2 cells were transfected with pEGFP-N1 and either pcDNA3-HA (Mock) or APIP-HA, and then incubated with serum-free medium for 24 h. After pre-treatment with vehicle (Mock) or 10 μM H-89 for 5 min, cells were incubated under normoxic (Nor) or hypoxic (Hypo) condition for 18 h (***P < 0.001, one-way ANOVA/Bonferroni, n = 3)