Effect of PTX on in vitro migration and actin polymerization. PTX blocks in vitro migration and actin polymerization of BM and FL c-kit⁺ cells. The c-kit⁺ cells from BM or 15-dpc FL cells were enriched as described in “Materials and methods” and incubated in SCF with or without PTX (2 hours or 20 hours for panel A, 20 hours for panels B-F). *P < .05 compared with control. (A) Chemokinetic and chemotactic (SDF-1) transwell migration of c-kit⁺ BM cells was assessed. SDF-1–induced chemotaxis was significantly attenuated after 2 hours of PTX incubation (middle), but migration was almost completely blocked after 20 hours of PTX incubation (right) (chemokinetic migration, light gray bars; chemotactic migration, dark gray bars). (B) Adhesion of c-kit⁺ BM cells to RetroNectin under laminar flow was identical in PTX-treated and control cells (control cells, ▦; PTX-treated cells, ▪). (C) SDF-1–induced actin polymerization was measured by staining F-actin with phalloidin. The rapid and transient SDF-1–induced actin polymerization in control cells was blocked in PTX-incubated c-kit⁺ BM cells (control cells, ▦; PTX-treated cells, ♦). (D) After a 10-second stimulation with SDF-1, control (i), but not PTX-treated (ii), cells showed increased phalloidin staining with a predominantly perinuclear pattern (DeltaVision; original magnification × 60). (E) Transwell migration of c-kit⁺ FL cells was tested as described in “Materials and methods.” Chemokinetic migration of c-kit⁺ FL cells was low. SDF-1–induced chemotaxis was completely inhibited by PTX (right) compared with control (left) (chemokinetic migration, light gray bars; chemotactic migration, dark gray bars). (F) SDF-1 induced actin polymerization in control, but not in PTX-treated, c-kit⁺ FL cells. The actin response of FL cells was slower than that of BM cells, where the maximum was reached after 10 seconds (control cells, ▦; PTX-treated cells, ▪). Error bars indicate SEM.