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

By Abderrahim El Guerrab (343565), Rabah Zegrour (343566), Carine-Christiane Nemlin (343567), Flavie Vigier (343568), Anne Cayre (70337), Frederique Penault-Llorca (4018373), Fabrice Rossignol (70338) and Yves-Jean Bignon (54433)

Abstract

<p>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% O<sub>2</sub> 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).</p

Topics: Physiology, Biotechnology, Chemistry, Cancer, gfp-p564, ca9-gfp, hypoxia-inducible
Year: 2013
DOI identifier: 10.1371/journal.pone.0025080.g003
OAI identifier: oai:figshare.com:article/402843
Provided by: FigShare
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