Deficiency of ROCK1 results in increased adhesion on fibronectin. (A) WT and ROCK1−/− BMMs (5 × 105) were subjected to an in vitro adhesion assay on fibronectin fragment CH-296. Representative images shown here are of adherent WT (left panel) and ROCK1−/− BMMs (right panel) on fibronectin at different time points. Results shown here are from 1 independent experiment. Similar results were obtained in 2 other independent experiments. Images were acquired through a Zeiss Axioskop 2 Plus microscope equipped with a Plan-Neofluar 20×/0.5 objective lens, and were captured with an Axiocam MRC-5 camera and Axiovision 4 software (all from Zeiss). (B) Quantitative adhesion was assessed by measuring absorbance at indicated times. Bar graph represents the optical density of adherent cells at 600 nm from 1 independent experiment. WT versus ROCK1−/−, *P < .05. Similar results were obtained in 3 other independent experiments (total n = 4). (C) Expression of β1 integrins on WT and ROCK1−/− BMMs. BMMs were stained with PE-conjugated anti-α4β1 or anti-α5β1 antibody and subjected to flow cytometric analysis. Top panel (solid histograms) represents the level of α5β1 expression on the surface of WT and ROCK1−/− BMMs; bottom panel (solid histograms) represents the level of α4β1 expression on the surface of WT and ROCK1−/− BMMs, whereas open histograms demonstrate the level of expression using an isotype control antibody in both panels. Percentage of α4β1 or α5β1 integrin expression on BMMs is shown in the top right quadrant of each histogram (total n = 4).
