TSP1 antagonizes NO-dependent alterations in F-actin and dephosphorylation of MLC in VSMCs. HAVSMCs plated on glass chamber slides were incubated in basal medium with 0.1% BSA (A-B) or 2.2 nM TSP1 (C-D) ± DEA/NO (10 μM). Cells were then fixed, permeabilized, and stained with Oregon Green-phalloidin to visualize F-actin. Photomicrographic images were acquired on a Nikon Eclipse E1000 microscope (Nikon, Melville, NY) using a Plan Apo objective lens. Low-magnification images were taken at 20× and a numeric aperture of 0.75; high-power images, at 40× and a numeric aperture of 0.95. No imaging medium or solution was used. A Cool Snap FX camera (Roberts Scientific, Tucson, AZ), IP Lab 3.5 software (Scanalytics, Fairfax, VA), and Photoshop C5 (Adobe Systems, San Jose, CA) were used for image acquisition and processing. Photomicrographs representative of 3 separate experiments are presented. Scale bar = 50 μm. HAVSMCs in 96-well plates were similarly treated and stained, and the fluorescence was signal quantified (E). *P < .05 versus BSA − NO, Student t test. #P < .05 versus BSA + NO, $P < .05 versus BSA − NO, 2-way ANOVA. &P < .05 versus S1P − NO, one-way ANOVA. Lysates of HAVSMCs in growth medium with 2% serum and treated with the indicated combinations of 100 nM S1P, 10 μM DEA/NO, and 2.2 nM TSP1 for 5 minutes were separated by SDS–polyacrylamide gel electrophoresis (PAGE) and analyzed by Western blot to determine the levels of MLC phosphorylation and total MLC (F). The blot shown is representative of 4 independent experiments. Results are presented as the mean ± SD.