Rosuvastatin and Migration of LDL-Loaded VSMC Figure 3. Rosuvastatin prevents MRLC dephosphorylation in VSMC exposed to LDL. (A) Representative 2-DE gel of the cytoskeleton/ membrane fraction. Arrows show spots identified as MYL9 (sarcomeric-MRLC), non-sarcomeric-MRLC (MRLC) and alkali-MLC (MLC). Enlarged images for the proteomic pattern of the non-sarcomeric MRLC isoforms (MRLC-2, spot 1–2 and MRLC-3 spots 3–4). Note that numbers 1–2 and 3–4 denote double spots with different pI of the non-sarcomeric MRLC isoforms. (B) Bar-diagrams for ratios between the intensity of the spot with lower pI and the correspondent with higher pI. Results given as mean6SEM (n = 3). P values for Man-Whitney test; only shown differences for P,0.05 (C) Western blot for phosporylated-MRLC (P-MRLC) and total MRLC in extracts of VSMC exposed to agLDL in the presence of different dose of rosuvastatin (2.5– 10 mM). C denotes the control group. Bar diagram represents quantitative values (mean6SEM) of 3 independent experiments. P values for Man- Whitney test. doi:10.1371/journal.pone.0038935.g003 regularly controlled by cell-staining with Herxheimer reagent (Sudan III and Sudan IV). In all experiments LDL-oxidation (before and after aggregation) was excluded by measuring thiobarbituric acid-reactive substances and by a monoclonal antibody 4E6-based ELISA (Mercodia) [18]. Cell migration and wound repair assay A double sided scrape-wound was made on confluent VSMC monolayers, as previously described [18]. Cell migration and wound repair were analyzed over a period of 12 hours (37uC) on 10% FCS-stimulated cells. Images were taken under a 610 lens, using an inverted microscope (Leica DMIRE2) attached to a video SPOT camera (Leica-DFC350FX), on 3 different fields along the linear scratch covering more than the 95% of the wound. The area free of cells in each field was calculated using ImageJ software, and values averaged. Measurements were made at time 0 and during cell migration (2, 4, 8 and 12 hours). Cell migration and wound repair were indirectly assessed by measuring the residual cell- denuded area. Values were expressed as a percentage of the cell- depleted area at time 0. PLoS ONE | www.plosone.org 4 June 2012 | Volume 7 | Issue 6 | e38935