Signaling pathways leading to SDF-1–induced CD9 expression. (A) Cord blood CD34⁺ cells, either untreated (IMDM or dimethylsulfoxide [DMSO]; ie, solvent control), or pretreated with AMD3100 (AMD; 10 μg/mL), pertussis toxin (PTX; 1 μg/mL), PD 98 059 (PD; 40μM), wortmannin (W; 50 nM), GF 109203X (GF; 2μM), chelerythrine chloride (CC; 5μM), AG490 (AG; 50μM), U-73 122 (U; 2.5μM), or D609 (D; 10μM) for 1 hour, were stimulated with or without SDF-1 (100 ng/mL) for 4 hours. CD9 and CXCR4 expressions were detected by flow cytometry (n = 4-8). Results are represented as the percentage changes in CD9⁺ or CXCR4⁺ cells after SDF-1 stimulation in the presence of each inhibitor. AMD and PTX were dissolved in IMDM, while other inhibitors were dissolved in DMSO. (B) Enriched CD34⁺ cells were left untreated (filled histogram) or stimulated with SDF-1 (200 ng/mL; open histogram) for 1 minute. (i) Phosphorylated Akt (pAkt) and (ii) phosphorylated ERK (pERK) levels were measured by flow cytometry. Representative histograms and the mean fluorescence intensity are shown. (C) Cells were stimulated with SDF-1 (200 ng/mL) for 1 minute after pretreatment with wortmannin (W; 50nM), chelerythrine chloride (CC; 5μM), AG490 (AG; 50μM) or U-73 122 (U; 2.5μM) for 1 hour. Phosphorylated ERK (p-ERK) level was detected by flow cytometry (n = 3). (D) CD34⁺ cells were cultured for 4 hours in the absence (DMSO) or presence of PKC activators PMA (200 ng/mL), MEZ (200 ng/mL), or IDB (200 ng/mL). Cell-surface CD9 and CXCR4 expressions were detected by flow cytometry (n = 3). (E) Proposed model for the signaling pathways of SDF-1–induced CD9 expression. Results are depicted as means ± SEM. *P ≤ .05; **P < .01.