in ice-cold physiological salt solution (PSS) containing (in mM) NaCl 137, KCl 5.6, NaH2PO4 0.4, Na2HPO4 0.4, NaHCO3 4.2, MgCl2 1, CaCl2 2.6, Hepes 10, and glucose 5 with pH adjusted to 7.4 with NaOH. The small arteries were cut into 5 mm-long pieces, and incubated at 37uC in low-Ca2+ PSS (0.1 mM CaCl2) containing 1 mg/ml albumin, 0.5 mg/ml papain, and 1 mg/ml dithioerythritol for 30 min, and for another 20 min in the Ca2+- free PSS containing 1 mg/ml albumin, 0.8 mg/ml collagenase, and 0.8 mg/ml hyaluronidase. Single cells were released by gentle trituration through a Pasteur pipette, stored in the nominally Ca2+-free PSS at 4uC, and used the same day. These primary SMCs were grown in phenol red-free Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin. The experiments were performed when the cells reached 70–80% confluence between passages 6 and 10. In all studies, cells were first incubated in the serum-free medium for 24 h and then 1% serum added together with different treatments, the media were changed every 2 days. Cell Viability Assay Cell viability was determined by the 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described previously [18]. In brief, equal numbers of cells were seeded into each well of 96-well plates for 24 h. After different treatments, MTT (final concentration, 5 mg/ml) was added to each well, and then the cells were incubated for 4 h at 37uC. The cell culture medium was removed, and dimethyl sulfoxide (200 ml/well) was added. The absorbance of formazan products at 570 nm was measured in a FLUOstar OPTIMA microplate spectrophotometer (BMG LABTEch, Offenburg, Germany). The cells incubated with control medium were considered 100% viable. Cell Proliferation Assay Cell proliferation was assessed by bromodeoxyuridine (BrdU) incorporation as described previously [8]. SMCs were plated at equal numbers of cells (2.06103 per well) in a 96-well plate for 24 h in the presence of 10% FBS. BrdU label was added into the culture media for another 24 h. Incorporated BrdU was detected following the procedures provided by the manufacturer (Calbio- chem, Gibbstown, NJ, USA). For the experiments with estrogen treatments the cells were first seeded in a 96-well plate for 24 h in media containing 10% FBS, and then changed to a serum-free medium for another 24 h. Afterwards, 1% FBS was added back together with estrogen (0.1–1000 nM) for 48 h following BrdU label for additional 24 h. Measurement of H2S Production H2S production rate was measured as described previously [19]. In brief, after different treatments, the cells were collected and homogenized in 50 mM ice-cold potassium phosphate buffer (pH 6.8). The flasks containing the reaction mixture (100 mM potassium phosphate buffer, 10 mM l-cysteine, 2 mM pyridoxal 5- phosphate, and 10% cell homogenates) and center wells contain- ing 0.5 ml 1% zinc acetate and a piece of filter paper (262.5 cm) were flushed with N2 gas and incubated at 37uC for 90 min. The reaction was stopped by adding 0.5 ml of 50% trichloroacetic acid, and the flasks were incubated at 37uC for another 60 min. The contents of the center wells were transferred to test tubes, each containing 3.5 ml of water. Then 0.5 ml of 20 mM N, N- dimethyl-p-phenylenediamine sulfate in 7.2 M HCl and 0.5 ml 30 mM FeCl3 in 1.2 M HCl was added. The absorbance of the resulting solution at 670 nm was measured 20 min later with a FLUOstar OPTIMA microplate spectrophotometer. Interaction of H2S and Estrogen on Proliferation Short Interfering RNA (siRNA) and Plasmid Transfection Transfection of SMCs by siRNA or plasmids (pCMV-Cyclin D1 and pEGFP-C1-ERa as well as corresponding control plasmid) was achieved by using the LipofectamineTM 2000 transfection agent from Invitrogen (Burlington, ON). In brief, SMCs were seeded at equal number of cells (2.06105 per plate) in 60 mm2 plates with the medium containing 10% FBS. The cells were plated to form 60–70% confluent monolayers for siRNA trans- fection, and 80–90% confluence for plasmid transfection. siRNA or plasmid and the transfection reagent complex were added to the serum-free medium for 4 h, and the transfection continued for another 48 h (for siRNA transfection) or 24 h (for plasmid transfection) in serum-containing regular medium. After that, the cells were collected for detection of protein expressions with western blotting analysis. Western Blotting Analysis Cells were harvested and lysed. Equal amounts of proteins were boiled and separated with SDS-PAGE and electrophoretically transferred to a nitrocellulose membrane, as described previously [20]. In each lane of a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, equal amounts of proteins were applied, electrophoresed and transferred to a polyvinylidene fluoride membrane. Membranes were blocked with Tris-buffered saline containing 5% non-fat milk at room temperature for 1 h, then incubated overnight at 4uC with primary antibody. The primary antibody dilutions were 1:1,000 for CSE, ERa, ERb and Cyclin D1, phosphorylated or total ERK, p38 MAPK, or JNK, and 1:10,000 for b-actin. The membrane was then washed three times with 16Tris-buffer saline-Tween 20 (TBST) buffer and incubated in TBST solution with horseradish peroxidase-conjugated second- ary antibody (diluted 1:5,000) for 1 h at room temperature on a shaker. Finally, the membrane was washed with TBST solution for 3 times. The immunoreactions were visualized with ECL and exposed to x-ray film (Kodak Scientific Imaging film, Kodak, Rochester, NY). Real-time PCR Analysis Total RNA was isolated using an RNeasy Mini Kit (Qiagen, Germantown, MD) and converted to cDNA with an iScriptTM cDNA Synthesis Kit (Bio-Rad, Hercules, CA). Real-time PCR was performed in an iCycler iQ5 apparatus (Bio-Rad) associated with the iCycler optical system software (version 3.1) using SYBR Green PCR Master Mix, as described previously [8]. The primers of ERa were 59-ATTCTGACAATCGACGCCAG-39 (forward) and 59-GTGCATTGGTTTGTAGCTGG-39 (reverse). The pri- mers for b-actin were 59-ATGGTGGGAATGGGTCAGAA-39 (forward) and 59-CTTTTCACGGTTGGCCTTAG-39 (reverse). The PCR conditions were as follows: denaturation at 95uC for 60 s, followed by 40 cycles of denaturation at 95uC for 15 s, annealing at 55uC for 15s, and extension at 72uC for 45 s. Relative mRNA quantification was calculated by using the arithmetic formula ‘‘2-DDCT’’, where DCT is the difference between the threshold cycle of a given target cDNA and an endogenous reference b-actin cDNA. Statistical Analysis All data were expressed as the mean 6 SE and represented at least three independent experiments. Statistical comparisons were made using student’s t-test or one-way ANOVA followed by a post hoc analysis (Tukey test) where applicable. Significance level was set at p,0.05. PLoS ONE | www.plosone.org 3 August 2012 | Volume 7 | Issue 8 | e41614