Figure 5
Figure 5. Tak779 inhibits R5 HIV-1–mediated PC-PLC activation and CCL2 secretion in MDMs. (A) Monocytes were seeded and cultured as described in “Methods.” At day 7, cells were treated with Tak779 (5 μM) for 30 minutes prior to R5 gp120 exposure (1 μg/mL) for 5 minutes. Some cultures were exposed to CCL4 (200 ng/mL) for 5 minutes. Cells were then lysed and PC-PLC enzymatic activity was measured in cell lysates as described in “Methods.” The relative contribution of PC-PLD was subtracted from the total fluorescence. Data are the means (± SD) of the results obtained with 3 different donors. (B) Monocytes were seeded and cultured as described in “Methods.” At day 7, cells were treated with Tak779 (5 μM) for 30 minutes prior to R5 gp120 exposure (1 μg/mL) for 5 minutes. Some cultures were exposed to CCL4 (200 ng/mL) for 5 minutes. Cells were then fixed, permeabilized, and stained with rabbit anti–PC-PLC Abs (detected in white) as described in “Methods.” PC-PLC expression and cellular localization were detected by CLSM analysis. The results from 1 representative experiment of 3 independently performed are shown. The bars, indicated in the lower panels, correspond to 20 μm. Right panels show a single cell of the corresponding left panels at higher magnification. (C) Monocytes were seeded and cultured as described in the footnote to Table 1. MDMs were treated with Tak779 (5 μM) or left untreated. After 30 minutes, some cultures were exposed to R5 gp120 (1 μg/mL) or CCL4 (200 ng/mL). After 24 hours of culture, supernatants were harvested and frozen before CCL2 determination. Data are the means (± SD) of the results obtained with 7 different donors. (D) Monocytes were seeded and cultured as described in the footnote to Table 1. MDMs were treated with Tak779 (5 μM) or left untreated. After 30 minutes, some cultures were exposed to R5 AT-2–inactivated viruses (17 ng HIVp24gag/106 cells). After 24 hours of culture, supernatants were harvested and frozen before CCL2 determination. Data are the means (± SD) of the results obtained with 3 different donors. *P < .05; **P < .005.

Tak779 inhibits R5 HIV-1–mediated PC-PLC activation and CCL2 secretion in MDMs. (A) Monocytes were seeded and cultured as described in “Methods.” At day 7, cells were treated with Tak779 (5 μM) for 30 minutes prior to R5 gp120 exposure (1 μg/mL) for 5 minutes. Some cultures were exposed to CCL4 (200 ng/mL) for 5 minutes. Cells were then lysed and PC-PLC enzymatic activity was measured in cell lysates as described in “Methods.” The relative contribution of PC-PLD was subtracted from the total fluorescence. Data are the means (± SD) of the results obtained with 3 different donors. (B) Monocytes were seeded and cultured as described in “Methods.” At day 7, cells were treated with Tak779 (5 μM) for 30 minutes prior to R5 gp120 exposure (1 μg/mL) for 5 minutes. Some cultures were exposed to CCL4 (200 ng/mL) for 5 minutes. Cells were then fixed, permeabilized, and stained with rabbit anti–PC-PLC Abs (detected in white) as described in “Methods.” PC-PLC expression and cellular localization were detected by CLSM analysis. The results from 1 representative experiment of 3 independently performed are shown. The bars, indicated in the lower panels, correspond to 20 μm. Right panels show a single cell of the corresponding left panels at higher magnification. (C) Monocytes were seeded and cultured as described in the footnote to Table 1. MDMs were treated with Tak779 (5 μM) or left untreated. After 30 minutes, some cultures were exposed to R5 gp120 (1 μg/mL) or CCL4 (200 ng/mL). After 24 hours of culture, supernatants were harvested and frozen before CCL2 determination. Data are the means (± SD) of the results obtained with 7 different donors. (D) Monocytes were seeded and cultured as described in the footnote to Table 1. MDMs were treated with Tak779 (5 μM) or left untreated. After 30 minutes, some cultures were exposed to R5 AT-2–inactivated viruses (17 ng HIVp24gag/106 cells). After 24 hours of culture, supernatants were harvested and frozen before CCL2 determination. Data are the means (± SD) of the results obtained with 3 different donors. *P < .05; **P < .005.

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