Impaired myelopoiesis in Fak^−/− mice. (A) Whole BM cells from WT and Fak^−/− mice were analyzed for the expression of myeloid cell marker Gr-1 and Mac-1 by flow cytometry. The percentage of Gr-1/Mac-1 high double-positive cells (R2) and Gr-1/Mac-1 low positive cells (R3) in the BM are indicated in the square of each dot blot. Shown are results from a representative experiment. (B) Bar graph represents the mean values of Gr-1/Mac-1 high double-positive cells in the BM of WT and Fak^−/− mice (n = 8; *P < .05). (C) Whole BM cells from WT and Fak^−/− mice were stained with Annexin V and 7-AAD and analyzed by flow cytometric analysis. Numbers in each quadrant represent the percentage of cells in various stages of apoptosis. (D) Quantitative analysis of the percentage of BM cells undergoing early apoptosis (Annexin V) in WT and Fak^−/− mice. Bars represent the mean of 3 independent experiments consisting of 3 pairs of mice of each genotype for each experiment (n = 9; *P < .05). (E) Reduced CFU-Cs in Fak^−/− BM. LDBM cells (1.5 × 10⁴) from WT and Fak^−/− mice were plated in a methylcellulose colony-forming assay in the presence of the indicated cytokines. Colonies were enumerated 7 days later. Bar graph shows the mean number of colonies from 3 independent mice of each genotype plated in triplicates. *P < .05, Fak^−/− versus WT. Similar results were obtained in 3 additional pairs of mice of each genotype. (F) WT and Fak^−/− progenitors were subjected to an in vitro migration assay in response to SDF-1 or SCF on FN. Briefly, LDBM cells isolated from WT and Fak^−/− mice were cultured in the presence of SCF, TPO, IL-6, and FLT-3 for 2 days. Cells (0.5 × 10⁶) were subjected to transwell migration assay for 5 hours at 37°C. After 5 hours of incubation at 37°C, nonmigrated cells in the upper chamber were removed with a cotton swab. The migrated cells attached to the bottom surface of the membrane were stained with 0.1% crystal violet, dissolved in 0.1M borate, pH 9.0, and 2% ethanol for 5 minutes at room temperature. Photomicrographs shown are progenitor cells migrated on FN in response to SDF-1 or SCF. (G) The number of migrated cells per membrane was counted in 10 random fields with an inverted microscope using 20× objective lens; cells migrated into the lower chamber in response to SDF-1 or SCF are counted using hemocytometer from replicates of 3 from 1 experiment using 3 mice of each genotype. *P < .05, Fak^−/− vs WT, mean ± SEM. (H) Primary erythroid progenitor proliferation was assessed by incorporation of radioactive thymidine in WT and Fak^−/− cells. Briefly, erythroid progenitor cells grown for 2 days were starved in Stem Pro 34 medium without any growth factors or supplements for 4 hours. Erythroid progenitor cells (5 × 10⁴) were placed in a 96-well plate in 200 μL complete medium either in the absence or in the presence of indicated concentration of EPO, SCF alone, or in combination. Cells were cultured for 48 hours and subsequently pulsed with 1.0 μCi (0.037 MBq) [³H] thymidine for 6 hours. Cells were harvested using an automated 96-well cell harvester, and thymidine incorporation was determined as cpm. Bar graph shows pooled data from 2 independent experiments performed in replicates of 4 using 4 mice per genotype per experiment. *P < .05, Fak^−/− vs WT, mean ± SEM. (I) Quantitative analysis of the percentage of primary erythroid cells undergoing early apoptosis (Annexin V) in WT and Fak^−/− cells. Cells were harvested after 4 days of culture and starved in absence of growth factors or supplements for 6 hours. Bar graph represents the mean of Annexin V–positive cells for 1 independent experiment performed in triplicates. *P < .05, Fak^−/− vs WT, mean ± SD. (J) Erythroid progenitor cells were cultured for 4 days and starved in absence of growth factors or supplements for 4 hours and stimulated with SCF and EPO at 37°C. Cell lysates (35 μg) were subjected to Western blot analysis using indicated antibodies.