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Figure 2 | BMC Medicine

Figure 2

From: The cytoprotective drug amifostine modifies both expression and activity of the pro-angiogenic factor VEGF-A

Figure 2

Amifostine upregulates vascular endothelial growth factor A (VEGF-A) mRNA production independently of hypoxia-inducible factor (HIF). MCF7 cells were incubated in complete medium with WR-1065 in the presence of aminoguanidine (AG) or were subjected to low levels of oxygen (3% 02,). (A) Amifostine treatment does not lead to any accumulation of HIF proteins. Cells were treated for 6 h under 20% of oxygen (20% O2) with or without 1 mM WR-1065; or low oxygen conditions (3% O2). HIF-1α and HIF-2α were detected by Western blot (Ku80 used as internal control). (B) Amifostine treatment does not lead to the induction of HIF-1 target genes. Cells were treated with WR-1065, in the presence of AG, for 16 h or exposed to low oxygen conditions (3% O2) for 16 h. Total mRNA were isolated and the expression of HK-2 and GLUT-1 genes were assessed by quantitative polymerase chain reaction Q-PCR); β-actin served as internal control. (C/D) Amifostine-mediated VEGF-A upregulation is not impaired by a HIF-1α.shRNA (small hairpin RNA). (C) Western blot analysis of HIF-1α protein levels in MCF7 cells transduced with RFP.shRNA (control) and HIF-1α.shRNA constructions. Cells were treated with 1 mM WR-1065 for 16 h (upper panel, "+"), or exposed to low oxygen conditions (3% O2) or classical conditions (20% O2) for 6 h (lower panel). Proteins were analysed by Western blot using specific antibodies for HIF-1α and for Ku80 (control of loading). (D) VEGF-A mRNA expression was assessed by Q-PCR in MCF7 cells transduced with lentiviral constructs expressing either HIF-1α.shRNA or control RFP.shRNA (grey and white bars, respectively). Cells were incubated for 16 h in the presence of WR-1065 or subjected to low oxygen conditions (3% O2). Expression of global VEGF-A mRNA in HIF-1α.shRNA-expressing cells was determined by Q-PCR and expressed in fold-change relative to untransduced-cells left untreated. Significant expression changes were determined by Student's paired tests.

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