Effect of 17β-estradiol treatment on Notch canonical target genes in HUVECs.

<p>(A) HUVECs were treated with 1 nM E2 or DMSO (V) for 24 hours under M4 experimental conditions (2% FBS overnight followed by 20% csFBS). Total RNA was extracted and qRT-PCR analysis of Hes1, Hey1, Hey2 and HeyL genes expression was performed. Relative changes in mRNA expression levels were calculated according to the 2^−ΔΔCt method using RPL13A as reference gene. Results are expressed as mean ± SEM of three independent experiments, each performed in triplicate. (B) HUVECs were treated with 5 µM DAPT for 24 hours under M4 experimental conditions (2% FBS overnight followed by 20% csFBS). Cell lysates were electrophoresed and immunoblotted with Notch1 (C-20), Notch2 (clone C651.6DbHN) and Notch4 (H-225) antibodies to detect the transmembrane form of Notch1 (Notch1TM), the active form of Notch2 (Notch2IC) and the active form of Notch4 (Notch4IC). β-actin antibody was used to ensure equal loading. Densitometric analysis of Western blot assay is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071440#pone.0071440.s006" target="_blank">Figure S6D</a>. (C) HUVECs were treated with 5 µM DAPT for 24 hours under M4 experimental conditions (2% FBS overnight followed by 20% csFBS). Total RNA was extracted and qRT-PCR analysis of Hes1, Hey1 and Hey2 genes expression was performed. Relative changes in mRNA expression levels were calculated according to the 2^−ΔΔCt method using RPL13A as reference gene. Results are expressed as mean ± SEM of three independent experiments, each performed in triplicate. **P<0.01, significantly different from the control.