Interaction of H2S and Estrogen on Proliferation Figure 2. The effect of H2S on ERa and ERb expression. A, NaHS up-regulated the protein expressions of ERa in both WT-SMCs and CSE-KO- SMCs. The cells were treated with NaHS at 100 mM for 24 h. * p,0.05 vs. WT-SMCs. B, NaHS up-regulated the expression of ERa mRNA in both WT- SMCs and CSE-KO-SMCs. The cells were treated with NaHS at 100 mM for 24 h. * p,0.05 vs. WT-SMCs. C, NaHS had no effect on the protein expressions of ERb in both WT-SMCs and CSE-KO-SMCs. D, NaHS potentiated the inhibitory effect of estrogen on the growth of WT-SMCs. After WT- SMCs were treated with 100 nM estrogen for 72 h in the presence or absence of 100 mM NaHS, cell viability was measured. * p,0.05 vs. control group; # p,0.05 vs. estrogen group. All data were from three independent experiments. doi:10.1371/journal.pone.0041614.g002 Results The Opposite Effects of Estrogen on the Growth of WT- SMCs and CSE-KO-SMCs Estrogen at 10 to 1000 nM significantly decreased cell viability of WT-SMCs in a dose-dependent manner. However, at 10 to 100 nM estrogen significantly increased cell viability of CSE-KO-SMCs (Figure 1A and 1B). Cell viability data was further confirmed by cell proliferation analysis (Figure 1C and 1D). At the concentrations higher than 100 nM, the stimulatory effect of estrogen on pro- liferation of CSE-KO-SMCs could not be detected (Figure 1D). Effect of H2S on ERa Activation The physiological functions of estrogen are mostly mediated by estrogen receptors [3,4]. Our results showed that both mRNA and protein expressions of ERa were significant higher in WT-SMCs than that in CSE-KO-SMCs, and treatment with 100 mM NaHS (a H2S donor) for 24 h up-regulated ERa mRNA and protein expressions in both WT-SMCs and CSE-KO-SMCs (p,0.05) (Figure 2A and 2B). We further found that ERb protein expression was not changed between WT-SMCs and CSE-KO-SMCs, and NaHS treatment had no effect on the expression of ERb in both cells (p.0.05) (Figure 2C). In addition, NaHS at 100 mM PLoS ONE | www.plosone.org 4 August 2012 | Volume 7 | Issue 8 | e41614