ASB2α regulates cell spreading and podosome formation in DCs. (A-B) ASB2α^−/− and ASB2α^+/+ BMDCs were harvested, serum arrested for 1 hour in suspension, plated on fibronectin-coated coverslips, and fixed after 30 minutes. Cell areas of at least 100 cells were measured. Dot plots show the overall distribution, and lines show the median values. The percentage of cells with an area inferior to 250 µm², comprising between 250 and 750 µm² and superior to 750µm², were calculated. The data show means and SEM. (Sample size: ^+/+ = 5; ^−/− = 7). (C-E) ASB2α^−/− and ASB2α^+/+ BMDCs were harvested; serum arrested for 1 hour in suspension; plated on glass coverslips; fixed after 30 minutes; stained for FLNa, vinculin, and phalloidin; and then imaged. Scale bar represents 20 μm. Diagrams depict the intensity of the fluorescence for each staining along lines drawn on the enlarged overlay images. Percentages of cells with no adhesive structures (-), with individual podosomes (shown in the enlarged images a and c), or podosomes organized as rosettes (shown in the enlarged image b) were calculated in ASB2α^−/− and ASB2α^+/+ BMDCs (C) and in FLNa-negative (FLNa⁻) or FLNa-positive (FLNa⁺) ASB2α^+/+ BMDCs (D). The data show means and SEM. (Sample size: ^+/+ = 5; ^−/− = 6). *P < .05; **P < .01; ***P < .001.