The involvement of PI3K, ERK1/2, and Rac in SDF-1–induced F-actin polymerization. (A) CD34⁺ cells from healthy donors were pretreated with 10 μM U0126, 10 μM LY294002, or 40 μM NSC23766 for 30 minutes and stimulated with 100 ng/mL SDF-1 for the indicated time. Cells were fixed and permeabilized, and F-actin was stained with 2.5 U/mL OregonGreen 514-Phalloidin. The fluorescence intensity was determined by FACS analysis and expressed as a percentage of that in unstimulated cells. The mean of 5 independent experiments is shown. (B) HL60 cells were transduced with mock empty vector, Rac1V12 or Rac1N17, and GFP positive cells were sorted by MoFlow. Transduced cells were stimulated with SDF-1 for the indicated times, after which polymerized actin was stained with Alexa Fluor 594-Phalloidin. The fluorescence intensity was determined by FACS analysis, and expressed as a percentage of that in unstimulated cells. The mean of 3 independent experiments is shown. Significant differences are indicated with an asterisk (P < .05). (C) Normal CD34⁺ cells (n = 3) were transduced with mock empty vector or Rac1N17. Cells were stimulated with 100 ng/mL SDF-1 for the indicated time. Cells were fixed and permeabilized, and F-actin was stained with 2.5 U/mL Alexa Fluor 594-Phalloidin. Phalloidin binding in GFP + cells (ie, transduced cells) was determined by FACS analysis and expressed as a percentage of that in unstimulated cells. (D) Schematic representation of the involvement of SDF-1–activated signaling pathways, and their cross-talk interactions, in migration and actin polymerization of normal CD34⁺ cells.