Fig. 7

Effect of PHD2 knockdown on reduction of normoxic HIF-1α protein by AA in metastatic melanoma. WM9 metastatic melanoma cells were transfected using non-targeting control siRNA or siGENOME SMARTpool siRNA against PHD2. a siRNA transfected cells were treated for 24 h with or without 100 μM AA under standard normoxic culture conditions. HIF-1α and PHD2 were analyzed by western blot, and normalized to β-actin. Knockdown of PHD2 caused an increase in stabilized HIF-1α protein, however, does not result in loss of effectiveness of AA to reduce accumulated HIF-1α. b qPCR analysis of PHD1, 2, and 3 isoforms in untreated WM9 metastatic melanoma cells, normalized to β-actin expression. PHD2 appears is the prevalent isoform, however the presences of PHD1 may contribute to the retained activity of AA following PHD2 selective knockdown. c, d Densitometry analysis of HIF-1α and PHD2 expression following PHD2 knockdown and AA treatment. siRNA experiments were repeated a minimum of 2 additional times with similar results