Figure 3.

miR-31/FIH-1 regulates glycogen in a HIF-1α- and hydroxylase-independent manner. A, B) qRT-PCR assessment of CA9 (A) and VEGF (B) levels in HCEKs transduced with FIH-1 or an empty vector for 3 d. C) No changes were noted in the relative luciferase activity of a HIF-1 reporter in FIH-1-transduced HCEKs. D, E) qRT-PCR assessment of CA9 (D) and VEGF (E) mRNA levels in HCEKs treated with DMOG or a vehicle control for 2 d, showing marked increase in CA9 and VEGF. F) DMOG, a hydroxylase inhibitor, markedly increased luciferase activity of HIF-1 reporter-transduced HCEKs. G, H) qRT-PCR assessment of CA9 (G) and VEGF (H) mRNA levels in HCEKs treated with antago-31 or an irrelevant antagomir for 48 h. I) No changes were noted in the relative luciferase activity of a HIF-1 reporter in HCEKs treated with antago-31 or an ir-antago. A–I) J) HCEKs transduced with LZRS, LZRS-FIH-1-cds, or LZRS dnFIH-1 for 3 d were stained with PAS with and without amylase to digest glycogen. K) Glycogen density was quantified using computer-assisted image analysis. Both FIH-1-cds and dnFIH-1 significantly decreased glycogen, as determined by Student's t test. Error bars = sd derived from 3 experiments.