Figure 4. US28 promotes HIF-1 target gene transcription and reprograms activation of Akt and PKM2 which are involved in proliferation, angiogenesis and glucose metabolism through a Gα_q/CaMKII/Akt/HIF-1 dependent fashion in fibroblasts and glioma cells.

A. HIF-1 target genes GAPDH, GLUT1 and VEGFA relative mRNA levels were determined by quantitative RT-PCR using RNA isolated from US28 or mock transfected NIH-3T3 cells and gene-specific primers. Significant differences in HIF-1 target gene expression are depicted by asterisks (* = P < 0.05, **** = P < 0.0001). B. Lysates were prepared from synchronized U251-iUS28 cells with/without 48 hours of doxycycline-induced US28 expression and treatment with signaling pathway inhibitors. SDS/PAGE Western blot analysis for phosphorylated Akt (Ser 473), total Akt and the loading control β-actin. Relative Akt activity is semi-quantified by pAkt/Akt ratios C. Lysates were prepared from synchronized, stably transfected NIH-3T3 or U251-iUS28 cells and phosphorylation status of PKM2 were assessed by Western blotting. Relative PKM2 phosphorylation is semi-quantified by pPKM2/PKM2 ratios D. Native lysates were prepared from synchronized US28 or mock NIH-3T3 and U251-iUS28 cells. For U251-iUS28, native lysates were prepared after 48 hours of US28 expression with/without vehicle, DMSO or UBO-QIC treatment. PKM2 protein was cross-linked using 0.25% formaldehyde/PBS solution (+), lysates without cross-linking treatment (−) were included as loading and total PKM2 expression controls. PKM2 and β-actin expression levels were assessed by Western blotting.