Validation of the GFP-P564 and Ca9-GFP hypoxia-inducible reporters.

Hypoxic stimuli induced HIF-1 alpha accumulation in MDA-MB-231 cells and increased fluorescence in the GFP-P564 and Ca9-GFP cell models. Cells were treated with 200 µM cobalt (Co), 200 µM DFO, or 1 mM dimethyloxalylglycine (DMOG) or exposed to 1% O₂ for 24 H (hx). Untreated cells were incubated under normoxic conditions (nx) and used as controls. (A) Fluorescence was evaluated by flow cytometry, and the signal significantly increased (p<0.05) under hypoxic conditions. DFO induced (B) time- and (C) dose-dependent fluorescence, as assessed by flow cytometry in GFP-P564 and Ca9-GFP cells. (D) Total HIF-1 alpha was quantified in whole MDA-MB-231 cells using an ELISA assay. (E) HIF-1 alpha protein levels in MDA-MB-231 cells were analyzed by western blot. (F) HIF-1 alpha DNA-binding activity was analyzed in MDA-MB-231 nuclear extracts using a 96-well ELISA assay; the absorbance was assessed at 450 nm. Each well was coated with a dsDNA sequence containing the HIF-1 alpha response element. The data represent three independent experiments (average mean ± SEM).