Materials and Methods Materials Estrogen (17b-estradiol) was from Sigma (St. Louis, MO). The anti-CSE antibody was purchased from Novus Biologicals (Littleton, CO). The anti-MAPK antibodies and different MAPK inhibitors were obtained from New England Biolabs (Camarillo, CA). Anti-cyclin D1 antibody was from Lab Vision Corporation (Fremont, CA). Anti-ERa, ERb antibodies and siRNA for ERa as well as control siRNA were obtained from Santa Cruz Bio- technology (Santa Cruz, CA). Cyclin D1 siRNA and control siRNA were purchased from Ambion (Austin, TX). The plasmids pCMV-Cyclin D1 and pEGFP-C1-ERa were from Addgene (Cambridge, MA). Horseradish peroxidase-conjugated goat anti- rabbit IgG antibody and goat anti-mouse IgG antibody were from Sigma. All other chemicals were from Sigma or New England Biolabs. Interaction of H2S and Estrogen on Proliferation Isolation and Culture of SMCs Twelve-week-old male CSE-KO offspring and age matched male WT littermates on C57BL/6J6129SvEv background were used to isolate primary SMCs from mesenteric arteries [17]. All animals were maintained on standard rodent chow and had free access to food and water. All animal experiments were conducted in compliance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996) and approved by the Animal Care Committees of Lakehead University, Canada. In brief, 12- week male WT and CSE-KO mice were generally anaesthetized by intraperitoneal injection with a mixture of 10 mg/kg xylazine (Bimeda-MTC Animal Health, CA) and 100 mg/kg ketamine hydrochloride (BIONICHE Animal Health, CA). When mice have lost muscle tone and blink reflexes, pupils become fixed and respiration is regular, the operation was carried out. Mice were killed by decapitation. Small mesenteric arteries below the second branch of the main mesenteric artery were dissected out and kept Figure 1. Inhibition of WT-SMC growth but promotion of CSE-KO-SMC growth by estrogen. WT-SMCs and CSE-KO-SMCs were treated with estrogen at the indicated concentrations for 72 h, and then cell viability and cell proliferation were measured. Cell viability of WT-SMCs (A) and CSE-KO-SMCs (B) were measured by MTT assay and cell proliferation of WT-SMCs (C) and CSE-KO-SMCs (D) were analyzed by BrdU incorporation assay. The control cells without estrogen treatment were considered as 100% viable. Data were from five independent experiments. * p,0.05 vs. control group. doi:10.1371/journal.pone.0041614.g001 PLoS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e41614