Genistein inhibits SDF-1-mediated chemotaxis and HIV infection of resting CD4 T cells. (A) Resting memory CD4 T cells were stimulated with SDF-1 (12.5 nM) for various times, stained with FITC-phalloidin, and then analyzed with flow cytometer. (B) Resting memory CD4 T cells were stimulated with HIV-1NL4-3 for various times and similarly stained with FITC-phalloidin and analyzed. (C) Resting memory CD4 T ells were pretreated with pertussis toxin (PTX, 100 ng/ml), genistein (3.7 μM), herbimycin (1 μM), 8-Br-cAMP (100 μM), 8-Br-cGMP (100 μM), or DMSO (1%, control) for 15–60 minutes, and then assayed for migration towards SDF-1 (12.5 nM). Results are expressed as the relative percentage of migrating cells. (D) Dosage-dependent inhibition of SDF-1-mediated chemotaxis by genistein. Experiments were carried out as described in (C) with memory T cells pretreated with different dosages of genistein. (E) Resting CD4 T cells were similarly pretreated with genistein, herbimycin, 8-Br-cAMP, 8-Br-cGMP, or DMSO, and infected with HIV-1NL4-3 for 2 hours in the continuous presence of these inhibitors. Following infection, cells were washed twice and then cultured in the absence of the inhibitors for 5 days. Cells were activated at day 5 with anti-CD3/CD28 magnetic beads (4 beads per cell), and viral replication was measured by p24 release. (F) Dosage-dependent inhibition of HIV-1 infection by genistein. Experiments were carried out as described in (E) with resting T cells pretreated with different dosages of genistein. (G) Effects of genistein on T cell activation. Resting CD4 T cells were treated with genistein at different dosages for 3 hours, washed twice, and then cultured in the absence of genistein as described in (E). Cells were similarly activated with anti-CD3/CD28 beads. At 24 hours after stimulation, cells were stained with PE-labeled monoclonal antibody against human CD25 or CD69, and then analyzed with flow cytometry.