Inhibition of PKC-ζ impairs cytoskeletal rearrangements and adhesion of CD34⁺ cells. (A) CB CD34⁺ cells, either untreated (Ctl) or preincubated with 10 μM PS-α/β or -ζ peptides, 10 μg/ml anti–VLA-4 or nonrelevant IgG, or 10 μM LFA-1 inhibitory or control peptides were subjected to adhesion assay to MS-5 cells. Data show average ± SD of 3 independent experiments; *P < 0.05. (B) Actin polymerization assay. CD34⁺ cells pretreated with chelerythrine chloride or with PS-α/β or -ζ peptides were stimulated with SDF-1 for the indicated times, and phalloidin-FITC fluorescence intensity was measured by flow cytometry. (C–G) SDF-1–induced cell polarization of CD34⁺ cells: untreated cells (C), SDF-1–treated cells (D), SDF-1/PS-ζ (E), and SDF-1/PS-α/β (F). Cell morphology and polymerized actin distribution were examined by phalloidin staining. Scale bar: 10 μm. Arrows indicate cells displaying highly polarized morphology in response to SDF-1. Cells similar to ones indicated by asterisks are shown in insets (original magnification, ×100). Quantification of the number of cells with elongated morphology (C, inset) and highly polarized morphology (D and F) from 1 representative experiment is shown (G). (H and I) Spreading and motility under laminar shear flow. G2 cells, control (H) or pretreated with PS-ζ peptides (10 μM) (I), were perfused on VCAM-1/SDF-1–coated plates under shear flow. Percentage of motile cells was determined by video analysis (Supplemental Videos 1 and 2). Original magnification, ×20. Arrows indicate protrusions formed in control cells (H), which are absent in cells treated with PS-ζ peptides (I).