TSA inhibits VEGFR2 in BCECs and HUVECs.

<p>Real-time PCR (A) and Western blot (B) were performed on BCECs (left panel) and HUVECs (right panel) treated with 0–0.7 μM TSA for 24h or 48 h. (A-B) TSA exerts a dose-dependent reduction effect on the mRNA and protein levels of VEGFR2. (C) Densitometry data for Western blot result of VEGFR2 normalized by GAPDH levels for BCECs (left panel) and HUVECs (right panel). (*: t test p<0.05, **: t test p<0.01) (D) HUVECs were treated with 0–0.7 μM TSA for 48 h, and then stimulated with 25 ng/mL of human recombinant VEGF for 10 min, followed by Western blot analysis (left panel). VEGF significantly induces the phosphorylation of VEGFR2, but the phosphorylation was attenuated by TSA at 0.7 μM, concomitant with a down-regulation of VEGFR2 total protein level. Densitometry data (right panel) for Western blot result of VEGFR2 and phospho-VEGFR2 normalized by GAPDH levels. (*: t test p<0.05) (E) ChIP assay was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120587#pone.0120587.g003" target="_blank">Fig. 3</a> on HUVECs treated with 0.5 μM TSA for 48 h. Released chromatin was then amplified by PCR using primers targeting VEGFR2 encompassing the region from 200 bp upstream of the transcription start site to 200 bp downstream of the transcription start site. Amplified chromatin was then run on a 1% agarose gel. Less promoter opening was found in TSA-treated cells than in untreated cells. Densitometry of ChIP assay result normalized by input levels. (*: t test p<0.05).