Increased F-actin in ROCK1^−/− BMM. (A) BMMs were seeded on a glass slide overnight. The next day, cells were fixed and stained with Alexa 488–phalloidin to detect F-actin shown in green. Representative confocal microscopy images demonstrate the expression of F-actin on WT, ROCK1^−/− unstimulated, and stimulated BMMs. Top panel represents F-actin content in green on WT and ROCK1^−/− BMMs unstimulated condition; bottom panel represents F-actin content in green on WT and ROCK1^−/− BMMs on stimulation with M-CSF. 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) Images were captured by a confocal laser-scanning microscope (Zeiss LSM 510), and F-actin content was quantified using ImageJ software. Quantification data are represented as F-actin content expressed as arbitrary units. Bar graph represents compiled data from 3 independent experiments in which 50 BMMs were randomly quantified from 10 different fields (n = 3, *P < .05). (C) BMMs were stained with Alexa 488–phalloidin to determine F-actin content and subjected to flow cytometric analysis. Bottom right quadrant represents the percentage of F-actin–positive WT (left panel) and ROCK1^−/− (right panel) BMMs. Data shown are from a representative experiment. Similar results were obtained in 3 other independent experiments. (D) Quantification of F-actin content in WT and ROCK1^−/− BMMs is represented as percentage of F-actin content from 3 experiments, shown as bar graph (total n = 4, *P < .05).