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Figure 3 | BMC Medicine

Figure 3

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

Figure 3

Amifostine activates the unfolded protein response-dependent signalling pathways. MCF7 and U87 cells were treated for increasing periods of time with 2 mM WR-1065 or 10 μg/mL Tunicamycin (Tun) in complete culture medium. Proteins and mRNA were collected at several time points, and protein and gene expression were assessed by Western-Blot and polymerase chain reaction (PCR) analyses. (A) Amifostine differentially up-regulates BIP, EDEM, GADD34 and CHOP genes in MCF7 cells. Quantitative-PCR profiles of BIP, EDEM, GADD34 and CHOP genes, in MCF7 cells treated with WR-1065. Results were normalized using α-tubulin mRNA and expressed as ratios between the treated [aminoguanidine (AG)+WR1065] and untreated (AG alone) conditions. The time zero of the experiment is set to 1. (B) Amifostine triggers eIF2α phosphorylation in MCF7 cells. Western-blot analysis of total (Tot.eIF2α) and phosphorylated eIF2α (P-eIF2α) in MCF7 cells treated or not with 2 mM WR-1065. A 1 h treatment with 2 mM dithiothreitol was used as a positive control for eIF2α phosphorylation. Total protein extracts were resolved on 12% SDS-PAGE, and protein levels were assessed using specific anti-eIF2α and anti-P-eIF2α antibodies. (C/D) Amifostine-induced vascular endothelial growth factor A (VEGF-A) up-regulation requires activating transcription factor 4 (ATF4). MCF7 cells were transduced with a small interfering (si)RNA directed against ATF4 (SiATF4, white bars) or with the same siRNA mutated in three nucleotides (SiMUT, grey bars), used as control;. Transduced cells were treated for 24 h with AG alone (CTL) or in combination with 2 mM WR-1065. (C) ATF4 gene expression was assessed by real-time reverse polymerase chain reaction (RT-PCR). (D) VEGF-A gene expression was assessed by real-time RT-PCR. ATF4 and VEGF-A mRNA expression levels in amifostine-treated cells are expressed as fold changes to the control condition (AG alone), which were set to 1 for SiATF4 and SiMUT. Error bars correspond to standard deviations for each triplicate determination.

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