164 Exp. Mol. Med. Vol. 44(2), 159-166, 2012 models such as atherosclerosis and vascular injury that are associated with phenotypic modulation of SMCs. Methods Materials Trypsin, α-minimum essential medium, fetal bovine serum, and Lipofectamine 2000 reagent were purchased from Invitrogen (Carlsbad, CA). Y27632 and simvastatin were purchased from Calbiochem (La Jolla, CA). D-erythro-SPC was purchased from Matreya (Pleasant Gap, PA). Anti-α-SMA antibody was purchased from Sigma-Aldrich (St. Louis, MO). Anti-phospho-Smad2 (Ser465/467) and anti-Smad2 antibodies were obtained from Cell Signaling Technology (Beverly, MA). Anti-RhoA antibody was pur- chased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was purchased from Millipore (Temecula, CA). Peroxidase-labeled secondary antibodies were obtained from Amersham Biosciences (Franklin Lakes, NJ). Cell culture Subcutaneous adipose tissue was obtained from elective surgeries with the patient’s consent as approved by the Institution Review Board of Pusan National University Hospital. To isolate hASCs, adipose tissues were washed at least three times with sterile PBS and treated with an equal volume of collagenase type I suspension [1 g/liter of Hank’s balanced salt solution (HBSS) buffer with 1% bo- vine serum albumin] for 60 min at 37oC with intermittent shaking. The floating adipocytes were separated from the stromal-vascular fraction by centrifugation at 300 × g for 5 min. The cell pellet was resuspended in α-minimum essen- tial medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin, and cells 2 were plated in tissue culture dishes at 3500 cells/cm . Primary hASCs were cultured for 4-5 days until they reached confluence and were defined as passage “0.” The passage number of hASCs used in these experiments was 3-10. Adipose tissues were collected from six different pa- tients, and six different hASC clones exhibited similar dif- ferentiation potentials toward osteogenic and adipogenic lineages as well as SMCs. Multipotent differentiation and proliferation capacities of hASCs remained consistent up until 10th passage. These six hASC clones exhibited SPC-induced α-SMA expression which could be inhibited by simvastatin treatment. They were positive for CD29, CD44, CD73, CD90, and CD105, whereas CD31 and CD45 were not expressed in hASCs (Supplemental Data Figure S1). Western blotting Confluent, serum-starved hASCs were treated with the ap- propriate conditions, washed with ice-cold PBS, and then lysed in lysis buffer (20 mM Tris-HCl, 1 mM EGTA, 1 mM EDTA, 10 mM NaCl, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, 30 mM sodium pyrophosphate, 25 mM β-glycerol phosphate, and 1% Triton X-100, pH 7.4). Lysates were resolved by SDS-PAGE, transferred onto a nitrocellulose membrane, and then stained with 0.1% Ponceau S solution (Sigma-Aldrich). After blocking with 5% nonfat milk, the membranes were immunoblotted with various antibodies, and the bound antibodies were vi- sualized with horseradish peroxidase-conjugated secon- dary antibodies using the enhanced chemiluminescence Western blotting system (ECL, Amersham Biosciences). Images were obtained using a LAS3000 image capture system (FUJIFILM, Japan). The immunoblots were quanti- fied by measuring the intensity of the protein bands with LAS3000 software. Immunoblot images were obtained at different exposure times, and images whose band in- tensities were within the linear range between the intensity increase and exposure time were selected for quantifica- tion of images. Measurement of membrane translocation of RhoA To determine the amounts of membrane-associated RhoA, membrane fractions were isolated as previously described (Negre-Aminou et al., 2001). Cells were made quiescent and stimulated as described in the figure legends. They were then washed in ice-cold PBS, lysed by incubation in hypotonic buffer (5 mM Tris-HCl, 5 mM NaCl, 1 mM CaCl2, 2 mM EGTA, 1 mM MgCl2, and 2 mM dithiothreitol, pH 7.0) containing a mixture of protease inhibitors. Membrane frac- tions were separated from cytosolic fractions by cen- trifugation (100,000 × g, 1 h at 4oC) of the lysates. Membrane fractions were prepared by resuspending the pellets in the hypotonic buffer. The lysates and membrane fractions were diluted in SDS-PAGE buffer and the protein levels of RhoA were determined by Western blotting. RhoA activation assay A commercial pull-down assay (Rho activation assay kit, Upstate) was used to measure the effect of SPC on RhoA activity in hASCs. The cells were treated washed twice with α-MEM and incubated for 24 h in fresh modified α-MEM without serum, SPC was added, and the cell sus- pension was centrifuged at the indicated time after SPC addition. After lysis of the cell pellet (lysis buffer, Upstate) and the activated RhoA was precipitated using a fusion protein consisting of GST and the Rho binding domain of Rhotekin, according to the manufacturer’s protocol. Protein concentrations were equalized between treatment groups prior to pull-down assay. Immunocytochemistry Cells were fixed in 4% paraformaldehyde in PBS for 15 min. For immunostaining, specimens were incubated with anti-α-SMA antibody for 2 h and Alexa Fluor 488-con- jugated anti-mouse secondary antibody (Invitrogen) for 1 h. The specimens were finally washed and mounted in Vectashield medium (Vector Laboratories) with 4',6-dia- midino-2-phenylindole (DAPI) for visualization of nuclei. Fluorescence images were collected using a Leica TCL-SP2 confocal microscope system (Leica Microsystems Heidelberg GmbH, Heidelberg, Germany).