D609 inhibits the R5 gp120-induced NF-kB p65 subunit nuclear translocation and CCL2 transcript accumulation. (A) Monocytes were seeded and cultured as described in “Methods.” At day 7, cells were treated with R5 gp120 (1 μg/mL) for 1 hour or left untreated. Some cultures were treated with D609 (50 μg/mL) for 30 minutes before gp120 exposure or with D609 alone. Cells were then fixed, permeabilized, and double-stained with rabbit anti-p65 Ab (detected in white) and DAPI (in blue) as described in “Methods.” p65 expression and cellular localization were detected by CLSM analysis. The results from 1 representative experiment of 3 independently performed are shown. The bar indicated in the lower panel corresponds to 20 μm. (B) Monocytes were seeded and cultured as described in “Methods.” MDMs were exposed to R5 gp120 (1 μg/mL) or left untreated. Some cultures were treated with D609 (50 μg/mL) or TPCK (10 μM) for 30 minutes before gp120 exposure or with D609 or TPCK alone. After 4 hours, total RNA was extracted, retrotranscribed, and amplified as described in “Methods.” The 2^−ΔΔCt values were calculated as described in “Methods.” Data are the means (± SD) of the results obtained with 3 different donors. **P < .005.