Deficiency of FAK in BMMs alters actin-based functions. (A) WT and Fak^−/− cells (2.5 × 10⁵) were subjected to an in vitro migration assay on FN, laminin, and collagen. 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) A quantitative assessment of the number of WT and Fak^−/− cells migrated through wells coated with FN, laminin, and collagen. Bars represent mean number of cells migrated ± standard deviation on various extracellular matrix proteins. Ten fields were scored in each experiment. Similar results were observed in 3 additional experiments (n = 3; *P < .05, WT vs Fak^−/−). (C) WT and Fak^−/− cells were cultured for 8 days in 24-well plates in the presence of 100 ng/mL M-CSF. An artificial wound was created in the monolayer using a pipet tip. Images were taken immediately and again at indicated time periods after creating the wound. Photomicrograph is from an independent experiment. (D) Bar graph shows quantitative analysis of the number of migrated cells in the wounded area. Data are from 1 representative experiment (n = 3; *P < .05 WT vs Fak^−/−). WT and Fak^−/− cells (5 × 10⁵) were subjected to an in vitro adhesion assay on FN, laminin, and collagen. Adhesion was assessed by measuring absorbance at indicated times on extracellular matrix proteins FN (E), laminin (F), and collagen (G). Bar graph represents the optical density of adherent cells at 600 nm. Data shown are from 1 representative experiment; *P < .05, WT vs Fak^−/−. Similar findings were observed in 3 independent mice. (H) Expression of integrins on WT and Fak^−/− cells. Cells were stained with PE-conjugated anti-α4β1 and PE-conjugated anti-α5β1 antibody and subjected to flow cytometric analysis. Top panel solid histograms indicate the level of α4β1 and α5β1 expression on the surface of WT cells and bottom panel solid histograms indicate the level of α4β1 and α5β1 expression on the surface of Fak^−/− cells. Open histograms demonstrate the level of expression using an isotype control antibody in both panels. Similar results were observed in 3 independent experiments. (I) WT and FAK-deficient cells grown in the presence of M-CSF for 3 days were starved of growth factors and stimulated on FN for indicated times. Lysates were subjected to Rac activity assay. Quantification of the level of active Rac is shown in bar graphs. Bottom panels show the level of active Rac (Rac GTP) and total Rac protein from 1 of 2 independent experiments performed.