SrfKO^H HSC/P cells exhibit defective adhesion, polarization, and motile responses to SDF-1. (A) Adhesion analysis. Srf⁺ and SrfKO^H fetal liver LSK cells were mixed 1:1 and seeded on fibronectin-coated substrate (i) or on monolayers of MBA-2.1 endothelial cells (ii), in the presence or absence of 100 ng/mL SDF-1. Solid symbols, Srf⁺; open symbols, SrfKO^H. See supplemental Figure 6D. (B) Morphology analysis. Cells were plated as in (A) on fibronectin-coated or MBA-2.1 endothelial cell monolayers, allowed to settle, and then stimulated for 45 minutes with SDF-1 before fixing and visualization with either Texas Red phalloidin (FN) or by prestaining with CFSE (MBA-2.1). Cell shape, defined as circularity = 4π (area/perimeter² ) was measured. A perfect circle has circularity 1.0, which decrease with increasing elongation. Solid symbols, Srf⁺; open symbols, SrfKO^H. See supplemental Figure 7A. (C) Transendothelial migration. CFSE-stained LSK cells were plated on SNARF-stained MBA-2.1 monolayers and treated as in (B). Panel Ci, cell locations were displayed by cumulative CFSE pixel intensity (LSKs, black lines) and [1-cumulative pixel intensity] (MBA-2.1, red line) relative to the membrane surface. Panel Cii, representative images: LSK cells in green and MBA-2.1 cells in semitransparent red. (D) Cell migration. LSK cells were plated on MBA2.1 cells and allowed to adhere for 20 minutes, and SDF-1 was added, followed by tracking for 20 minutes. The rose plots map the movement of each cell during the course of the experiment relative to its position at time zero. Data from 243 Srf⁺ and 228 SrfKO^H LSK cells tracked in 3 independent experiments are summarized.