Fig. 6: miR-3085-3p mediates HIF2α-dependent PKA Cα regulation in beige adipocytes.

a Prkaca 3′UTR sequence and predicted miR-3085-3p binding site. b Luciferase reporter activities of WT (n = 4) and mutant (n = 4) Prkaca 3′UTR upon con or miR-3085-3p transfection. c Upstream region of miR-3085-3p and hypoxia-response element (HRE) sites. d ChIP-qPCR analysis of BAC cells upon Admock (n = 5) or AdHIF2α (n = 5) infection. e miR-3085-3p level in iWAT from WT (TN; n = 5, Cold; n = 6) and HIF2α AKO (TN; n = 5, Cold; n = 6) mice upon TN or cold exposure (3 days). f miR-3085-3p level of iWAT from WT (n = 6), HIF1α AKO (n = 4), HIF2α AKO (n = 5), and HIF1/2α DKO (n = 5) mice upon cold exposure (3 days). g miR-3085-3p and h Prkaca levels in iWAT from vehicle- (n = 5) and PT2385- (n = 5) administered mice upon cold exposure (3 days). i mRNA levels in beige adipocytes from WT (n = 4) and HIF2α AKO (n = 4) mice upon con or miR-3085-3p mimic transfection with ISO (5 μM, 4 h). j OCRs and quantification in beige adipocytes from WT (n = 8) and HIF2α AKO (n = 8) mice upon con or miR-3085-3p mimic transfection. k mRNA levels in beige adipocytes infected with Admock (n = 4) or AdHIF2α (n = 4) upon con or miR-3085-3p inhibitor transfection with ISO (5 μM, 4 h). l OCRs and quantification in beige adipocytes infected with Admock (n = 10) or AdHIF2α (n = 10) upon con or miR-3085-3p inhibitor transfection. m Experimental scheme of miRNA mimic injection. n Representative images of H&E staining of iWAT from WT and HIF2α AKO mice with con or miR-3085-3p mimic injection upon cold exposure (3 days). Scale bars, 50 μm. o Western blot analysis of PKA Cα and UCP1 in iWAT from WT and HIF2α AKO mice with con or miR-3085-3p mimic injection upon cold exposure (3 days). Data were expressed as the mean ± SEM by two-tailed unpaired Student t-tests in (g, h), one-way ANOVA in (f), or two-way ANOVA in (b, d, e, i–l) followed by Holm–Sidak’s multiple comparisons test. Oligo. oligomycin, ISO isoproterenol, Rot. rotenone, AA antimycin A.