Fig. 3. BBI increases CC chemokines (ligands of HIV entry co-receptor CCR5).

Macrophages were treated with/without BBI at indicated concentrations for 6 h (mRNA) or 24 h (protein). Cellular RNA was collected and subjected to real time RT PCR for the genes indicated and GAPDH RNA (A–C). The data are expressed as RNA relative (fold) for RANTES, MIP-1α and MIP-1β to untreated control, which is defined as 1.0. Data are shown as mean±SD for three independent experiments. (D–F) RANTES, MIP-1α and MIP-1β proteins were analyzed by ELISA with the specific kits according to the manufacturer’s instructions. Data are shown as mean±SD for three independent experiments. (G) Macrophages were treated with/without BBI (100 μg/ml) for 4 h, and then washed with PBS. The cells were cultured with fresh medium for additional 20 h. The supernatant was collected as BBI-conditioned medium. BBI-conditioned medium was incubated with/without neutralization antibodies to RANTES, MIP-1α, MP-1β (CC chemokines) (20 μg/ml, respectively) for 1 h prior to HIV-1 (Jago) infection of macrophaegs. IgG antibody was used as the control. Cell-free supernatant was collected on day 5 for HIV Gag gene expression by the real time PCR. Data are shown as mean±SD for three independent experiments. (*P<0.05, **P<0.01, ***P<0.001 when performing Student’s t-test).