| Pathway description: |
Genes upregulated at 3% O2 are listed in Supplementary Table S2. The data revealed upregulation of key Notch mediators among others. RT-qPCR confirmed gene expression stimulation of key genes: Notch2, STAT6, and Jagged 1. Akt may therefore play a key role in promoting self-renewal, and thus we assessed whether oxygen tension affects Akt signaling. We incubated cells at 3% and 21% O2 for 30 and 60?min and observed strong Akt activation (phospho-Akt) by low oxygen after a 30-min transient exposure, which then returned to basal levels. Total Akt was unaffected .Since we observed 3% O2 stimulation of Lrp5 and frizzled 1 and 2 , we further examined canonical Wnt pathway activation. Western blot analysis of nuclear and cytoplasmic extracts of MIAMI cells demonstrated that 3% oxygen stimulated cytoplasmic β-catenin (1.5-fold) and, to a larger extent, nuclear β-catenin . Subsequently, we size separated, blotted, and probed the immunoprecipitated proteins with β-catenin antibody. Samples from cells at 3% O2 displayed more than twofold greater association between β-catenin and TCF4 versus 21% O2. we examined downstream targets of canonical Wnt activation. The cell cycle protein and direct Wnt target, Cyclin D1, was increased by 3% O2 about 2.2-fold in the cytoplasm and by more than 80-fold in the nucleus . |