Wnt/β-catenin signaling down-regulation is both dependent and independent of HIF expression.

A, Levels of active β-catenin were determined by western blot at different hypoxia time points for each of the shRNA in MNNG/HOS cells. Overall levels of active β-catenin decreased regardless of HIF expression, although the magnitude of decrease was not equal across all lines. Active and total β-catenin protein levels were decreased at the 72 hour time point. Actin was the loading control. B, Decreased Wnt/β-catenin signaling activity under hypoxia was confirmed by measuring axin2 mRNA levels via qrt-PCR in MNNG/HOS cells. Hypoxia (72 hour, 0.5% O2) resulted in decreased axin2 mRNA expression relative to normoxia. C, When analyzed relative to the shnon hypoxia mRNA, axin2 mRNA was increased in the shHIF-1α MNNG/HOS cell lines. Asterisks indicate statistical significance (*p<0.05, **p<0.01, ***p<0.001). shNON: non-targeting shRNA; HIF shRNAs used are indicated in parentheses.