Rosuvastatin and Migration of LDL-Loaded VSMC Figure 4. Rosuvastatin increases RhoA at the leading edge of migrating cells. Confocal microscopy for RhoA antigen labelling (green) in migrating cells (FCS-stimulated), 4 hours after the wound scratch. Immunocytology was performed in permeabilized cells. Red signal refers to F-actin (Alexa-Fluor 633 conjugated phalloidin). (A) Representative photomicrographs of RhoA labelling at the wound-border of control cells and agLDL- treated cells. (B) Effect of 10 mM rosuvastatin on RhoA localization at the leading-edge of control- and agLDL-VSMC. (C) Bar diagrams show quantitative levels (mean6SEM) of RhoA labelling over an area 15 mm wide, along the migrating edge (limited by dotted line in A). Intensity labelling signal was calculated using the LASAF Leica software and expressed in intensity/mm2 (results from 3 independent experiments). p,0.05 for (*) control+rosuvastatin vs control and (**) agLDL+rosuvastatin vs agLDL. doi:10.1371/journal.pone.0038935.g004 Cell Proliferation Arrested VSMC were incubated for 24 hours with 10 mM rosuvastatin and 100 mg/mL nLDL or agLDL in medium containing 0.5 mCi/mL of [3H]thymidine (Amersham) and cell proliferation was determined as previously described [41]. Cell adhesion assay Subconfluent VSMC, treated with/without rosuvastatin in the presence/absence of agLDL (16 hours), were harvested and resuspended in 5% FCS-M199 medium. Afterwards, 16105 cells were seeded on FCS-coated glass bottom dishes and further incubated for 3 hours in the presence/absence agLDL, without/ with rosuvastatin +/2 Y27632. After 3 hours, the attached cells were fixed with 4% paraformaldehyde for confocal microscopy studies or released by trypsination, stained with trypan blue for determination of cell viability and counted in a Neubauer- chamber. Experiments were performed in replicates and mean values were calculated. Protein extraction and proteomic analysis Frozen cell pellets were sequentially extracted in tris- and urea buffers to separate the cytosolic and membrane/cytoskeleton- enriched protein fractions [18]. Total cell lysates were made in RIPA-buffer. (50 mM Tris HCl pH 8; 150 mM NaCl; 1% NP-40; 0.5% sodium Deoxycholate; 0.1% SDS). Protein concentration was measured with 2D-Quant Kit (GE-Healthcare), as indicated by the manufacturer. For the proteomic analysis, sample contaminants were removed (2D-CleanUp Kit, Amersham) and proteins (120 mg) separated by 2-dimensional electrophoresis (2-DE) as previously described [18– 19]. Protein spots in the gels were labelled by fluorescence (Flamingo labelling, BioRad), scanned (Typhoon, GE-Healthcare), and analysed for differences in the protein patterns between groups with PD-Quest 8.0.1 software (8.0.1, Bio-Rad) [18–19]. Protein identification was performed by matrix-assisted laser desorption/ionisation-time of flight (MALDI-ToF) mass spectrom- etry (Ettan MALDI-ToF Pro, GE Healthcare) as previously described by our group [18]. Western blot analysis Protein antigen levels in cytosol and membrane fractions were analyzed by western blot, as described previously [18–19] using following primary antibodies: monoclonal anti-MRLC (Abcam, dilution:1/500), polyclonal anti-phosphorylated-MRLC (Abcam, dilution:1/500), monoclonal anti-RhoA (Santa Cruz, diution:1/ 500), polyclonal anti-p160ROCK (Santa Cruz, dil:1/500), mono- clonal anti-phosphorylated-PAK1 (Abcam, dilution:1/10000), and monoclonal anti-b-actin (Abcam, dilution:1/5000). Western blot bands were detected by chemiluminescence using a peroxidase enzymatic reaction (Supersignal, Pierce) and quantified with a PLoS ONE | www.plosone.org 5 June 2012 | Volume 7 | Issue 6 | e38935