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. Author manuscript; available in PMC: 2017 Aug 3.
Published in final edited form as: Sci Signal. 2016 May 31;9(430):ra56. doi: 10.1126/scisignal.aaf0583

Fig. 2. PKA increases HIF-1α protein abundance and transcriptional activity.

Fig. 2

(A and B) Immunoblot assays were performed using lysates from: primary human left-ventricle cardiomyocytes (CMs) exposed to vehicle (V), isoproterenol (ISO), or isoproterenol + phenylephrine (ISO/PE) (A; n = 2 biological replicates, as shown); or HeLa cells exposed to V, forskolin (F), H89 (H), or forskolin + H89 (F/H) at 20% or 1% O2 (B; representative of n = 3 independent experiments). (C) HeLa cells were co-transfected with p2.1 and pSV-Renilla, exposed to V, F, H, F/H, or PKI at 20% or 1% O2 and the ratio of firefly:Renilla luciferase was determined (HIF luciferase activity; mean ± SD, n = 9 biological replicates from 3 independent experiments). *p < 0.05 compared to V at same % O2; #p < 0.05 compared to forskolin at same % O2. (D) HeLa cells were co-transfected with p2.1, pSV-Renilla and vector encoding red fluorescent protein (RFP), RFP-Ca fusion protein, or HA-tagged Cb, exposed to 20% or 1% O2, and HIF luciferase activity was determined (mean ± SD, n = 9 biological replicates from 3 independent experiments). *p < 0.05 compared to RFP at the same % O2. (E) Immunoblot assays were performed using lysates of HeLa cells expressing RFP or RFP-Ca and exposed to 20% or 1% O2 (n = 3 independent experiments). (F and G) HIF-dependent luciferase reporter assays (F; mean ± SD, n = 9 biological replicates from 3 independent experiments; *p < 0.05 compared to SC at same % O2) and immunoblot assays (G; n = 3 independent experiments) were performed using lysates of HeLa cells expressing a scrambled control shRNA (SC) or either of two shRNAs targeting Ca (shCa-1 or shCa-5) and were exposed to 20% or 1% O2. (H) HIF-1α mRNA abundance was determined by RT-qPCR in parental HeLa cells (left panel), SC, shCa-1 and shCa-5 HeLa subclones (middle panel) or NRCMs (right panel), which were exposed to 20% or 1% O2 (mean ± SD, n = 6 biological replicates from 3 independent experiments; none of the differences were statistically significant). Data in C, D, F and H are graphed as fold change from respective controls at 20% O2. The Mann-Whitney U test was used to assess statistical significance for all data in this figure.