Interaction of H2S and Estrogen on Proliferation Figure 4. Estrogen altered the expression of cyclin D1. Estrogen reduced the expression of cyclin D1 in WT-SMCs (A) but up-regulated it in CSE-KO-SMCs (B). The cells were treated with 100 nM estrogen for 72 h. * p,0.05. C, Knockdown of ERa reversed estrogen-decreased expression of cyclin D1 in WT-SMCs. After transfection with siRNA ERa or siRNA C at 50 nM for 12 h, the cells were incubated with 100 nM estrogen for another 36 h. * p,0.05 vs. siRNA C group; # p,0.05 vs. siRNA C or siRNA C+estrogen group. D, Overexpression of ERa reversed estrogen-increased expression of cyclin D1 in CSE-KO-SMCs. After transfection with pEGFP-C1-ERa plasmid or control plasmid for 24 h, the cells were incubated with 100 nM estrogen for another 48 h. * p,0.05 vs. control vector group; # p,0.05 vs. control vector or control vector+estrogen group. All data were from three independent experiments. doi:10.1371/journal.pone.0041614.g004 PLoS ONE | www.plosone.org 7 August 2012 | Volume 7 | Issue 8 | e41614