Rescue of estrogen-dependent repression of miR34a by HDACs inhibitors.

<p>A) C27IM cells were treated with estradiol (E2) for 3 hours in the presence or absence of inhibitors of deacetylases (TSA, MS275, MC1568), or the SIRT1 inhibitor Sirtinol, or the SIRT1 activator Resveratrol, added 30 minutes before the hormone. Levels of pri-miR-34a (left) and mature-miR34a (right) were analysed by qRT-PCR. Data represent the mean ± SEM of 4 experiments. *p<0,05 B) C27IM were transfected with the eNOS dominant negative mutant (S1177A) or empty vector for 48h in presence or absence of E2 added 1h prior to harvesting. Levels of pri-miR-34a were analysed by qRT-PCR. Data represent the mean ± SEM of 3 experiments. *p<0,05 C) C27IM (left), LNCaP cells (middle) and HUVEC (right) were transfected with the eNOS dominant negative mutant (S1177A) or empty vector for 48 h in presence or absence of E2 added 1 h prior to harvesting. SIRT1 levels were assessed by western blot. Hsp70 served as loading control. Ratio +/−E2 of SIRT1 level normalized with Hsp70 are indicated. D, E) Confocal analysis of eNOS and HDAC4 (D) and HDAC5 (E) in the presence or absence of E2 as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062522#pone-0062522-g004" target="_blank">Figure 4</a>. F) Dynamic recruitment of class I (HDAC3), class II (HDAC4) deacetylases or SIRT1 analyzed by ChIP in the regulatory region of pri-miR34a in the presence or absence of E2. Arrows represent primers for qPCR as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062522#pone-0062522-g003" target="_blank">Figure 3</a>. *p<0,05 SIRT1 E2 vs SIRT1 NT; ^#p<0,05 HDAC3 E2 vs HDAC3 NT; §HDAC4 E2 vs HDAC 4 NT.