LRP1 Controls Vascular PI3K Figure 5. Primary SMC Migration. (A) Transwell migration assay. 30,000 SMCs of the indicated genotypes were added to the top compartment of a Boyden chamber. After 6 hours of incubation, the transwell membrane was fixed and stained with hematoxylin. Cells that had migrated through the holes on the membrane to the bottom face were counted. Scale bar, 50 mm. (B) Scratch assay. 300,000 SMCs of the indicated genotypes were seeded into 60 mm culture dishes in a medium containing 10 mg/ml mitomicin C to prevent cell proliferation and allowed to adhere overnight. The next day, part of the dish was denuded by scratching along a straight line (indicated by a black line behind). Cells were then allowed to migrate into the denuded area for 24 hours prior to fixation and quantification. Scale bar, 50 mm. (C, D) Statistical analyses of the Transwell and Scratch migration assays. Results are represented as mean6SD. *** p,0.001. (E) PDGF-BB chemotaxis assay. 10,000 SMCs of the indicated genotypes were added to the top compartment of the Boyden chamber. 10 ng/ml PDGF-BB was added to the lower chamber of the well. After 6 hours of incubation, the transwell membrane was fixed and stained with hematoxylin. The cells that had migrated through the holes on the membrane to the bottom face were counted. Scale bar, 50 mm. (F) Statistical analysis of the PDGF-BB chemotaxis assay. Results are represented as mean6SD. ** p,0.01, *** p,0.001. n = 5 for all assays. doi:10.1371/journal.pone.0006922.g005 PLoS ONE | www.plosone.org 7 September 2009 | Volume 4 | Issue 9 | e6922