Rosuvastatin and Migration of LDL-Loaded VSMC Figure 5. Increased levels of RhoA-GTP in cytosol of human VSMC exposed to agLDL in the presence of rosuvastatin. (A) Antigen (total RhoA) and active RhoA (RhoA-GTP) levels in total cell lysates and cytosol extracts of VSMC treated without/with rosuvastatin in absence/presence of nLDL and agLDL for 24 hours. Levels of active RhoA were determined by a pull down assay with GST-rothekin. Note the strong band for active RhoA in the cytosol of rosuvastatin treated cells. (B) Total and active RhoA levels in the cytosol fraction of VSMC treated without and with rosuvastatin +/210 mM geranyl-geraniol pyrophosphate (GGPP), in the absence and presence of nLDL and agLDL for 24 hours. (C) Western blot analysis for ROCK antigen levels in cytosol extracts of cells exposed for 24 hours to agLDL in the absence and presence of rosuvastatin. C denotes the control non treated group. doi:10.1371/journal.pone.0038935.g005 ChemiDocTM XRS system using the Quantity-One 1-D analysis software (Bio-Rad). Immunofluorescence and confocal microscopy Cells were fixed with 4% paraformaldehyde, permeabilized (0.5% Tween-PBS), and blocked with 1% bovine serum albumin (BSA). P-MRLC was detected with a rabbit polyclonal antibody (Santa Cruz) and RhoA with a monoclonal-antibody (Santa Cruz). The secondary antibodies were Alexa Fluor-488 conjugated anti- rabbit or anti-mouse antibodies (Molecular Probes dilution 1/100). Negative controls of primary and secondary antibody staining were run with each set of experiments. F-actin was stained with Alexa Fluor-633 phalloidin (Molecular Probe) and nuclear Figure 6. Rosuvastatin induced MRLC-phosphorylation in agLDL-VSMC is dependent on the activity of the RhoA/ROCK signaling pathway. VSMC incubated without/with agLDL, in the absence/presence of rosuvastatin analyzed by western blot for (A) P- MRLC in agLDL-VSMC incubated with rosuvastatin in the absence/ presence of inhibitors for MLC-Phosphatase (Y27632) and MLCK (ML9). Bar diagram expresses mean6SEM of 3 independent experiments. *P,0.05 (Mann Whitney test) vs agLDL. # P,0.05 (Mann Whitney test) vs agLDL+rosuvastatin group. (B) phosphorylated PAK (p21-activated kinase) levels in VSMC exposed to agLDL in the absence/presence of rosuvastatin. Bar diagram represent the mean6SEM of 3 independent experiments. P,0.05 (Mann Whitney test), * vs control group. doi:10.1371/journal.pone.0038935.g006 counterstain with Hoechst (Molecular Probes). Labelled cells were examined in a Leica inverted fluorescence confocal microscope (Leica TCS SP2-AOBS). Fluorescent images were acquired in a scan format of 102461024 pixels at intervals of 0,1 mm (20 slides) and processed with the TCS-AOBS software (Leica). Maximal intensity projection values were calculated using the LASAF Leica 2 Software and given as AU/mm . RhoA Activity Assay Levels of active RhoA were measured in cytosol extracts and in total cell lysate with a RhoA activation assay kit (BK036, Cytoskeleton) based in a pull-down technique, following the manufacturer’s instructions. Briefly, VSMC were starved 24 hours PLoS ONE | www.plosone.org 6 June 2012 | Volume 7 | Issue 6 | e38935