Vanin-1 in Vascular Remodeling Disease Figure 3. Relationships between PPARc expression, GSH content, and vanin-1 in arteries and cultured SMCs. A–B, We treated cultured aortic SMCs with diamide (5 mM) and/or PDGF (10 ng/ml) for 48 h and PPARc expression was analyzed by Western blot, and densitometry. P,0.05 for # Vnn12/2 control vs. WT control SMCs; *Vnn12/2 control vs. diamide treated SMCs; **WT SMCs vs. PDGF and diamide treated SMCs, respectively. C– D, SMCs were transfected with PPARc siRNA, and then PPARc expression analyzed by Western blot. *P,0.05 for Vnn12/2 vs. WT SMCs, # Vnn12/2 control vs. PPARc siRNA, **PPARc siRNA WT SMCs. E, WT and Vnn12/2 SMCs were treated with cysteamine (500 ng/ml) or BSO (1 mM), and GSH content determined after deproteinization. Data pooled from 3 experiments done in triplicate. # P,0.05 Vnn12/2 vs. WT SMCs, *control vs. PPARc siRNA,**control vs. cysteamine in both Vnn12/2 and WT SMCs, L control vs. BSO treatment in both Vnn12/2 and WT SMCs. In Panel F, WT and Vnn12/ 2 SMCs were treated with diamide (5 mM) or PDGF 10 ng/ml) for 24 h, and cell proliferation compared. *P,0.05 vs. control. G, SMC proliferation using PPARc siRNA knockdown. Data are mean 6 SD of 3 independent experiments. *P,0.05 control vs. PPARc siRNA, **PPARc siRNA vs. PPARc siRNA + PDGF in WT and Vnn12/2 SMCs. doi:10.1371/journal.pone.0039106.g003 SDS-PAGE and Western Blotting Aortic tissue and SMCs were lysed in RIPA buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mM sodium fluoride,1 mMsodium orthovandate, 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, 10 mg/ml leupeptin, 1 mg/ml aprotinin) and protein extracts separated by 10% SDS-PAGE, and transferred to Immobilon-P membranes. After blocking, blots were incubated with primary antibodies overnight at 4uC, and detection of antigen using secondary antibody conjugated with horseradish peroxidase, and enhanced chemiluminescence, as well as scanning densitom- etry, were done as described [38]. Pantetheinase Activity Assay The substrate, pantothenate–AMC was chemically synthesized using b-alanine 7- amido-4-methylcoumarin trifluoroacetic acid (TFA) salt (H- b -Ala-AMC.TFA, 36 mg, 1eq) and R-(-)- pantolactone [40]. To assay pantetheinase activity, we used pantothenate–AMC as substrate at 37uC for 30 min, with the hydrolysis catalyzed by vanin-1 yielding pantothenic acid and detectable free fluorescent AMC (excitation 340 nm, emission Figure 4. Vanin-1 mediates SMC proliferation in response to PDGF. Vanin-1 was expressed in Vnn12/2 SMCs by transfection using pCMV- VNN1 expression vector. A, Pantetheinase activity was measured in Vnn12/2 SMCs after transfection and B, Western blot analysis confirmed vanin-1 expression after transfection. C, Following pCMV-VNN1 or empty vector transfection, SMCs were treated with PDGF (10 ng/ml) for 24 h, and proliferation measured. Data are mean 6 SD of 3 independent experiments. *P,0.05 Vnn12/2 SMCs control vs. Vnn12/2 + pCMV-VNN1+ PDGF. doi:10.1371/journal.pone.0039106.g004 PLoS ONE | www.plosone.org 7 June 2012 | Volume 7 | Issue 6 | e39106