FIG 8.

Acetylsalicylic acid (ASA) is unable to inhibit reactivation from latency by HMBA plus bryostatin-1 in a CD4⁺ T_CM cell HIV-1 latency model. (A) Schematic of HIV-1 reactivation from the CD4⁺ T_CM cell latency model experiment (O/N, overnight). (B) ASA only mildly interferes with HIV-1 reactivation from latency obtained with HMBA plus bryostatin-1 treatment of the CD4⁺ T_CM cell latency model. For each experimental point, 5 × 10⁵ CD4⁺ T_CM cells latently infected with luciferase HIV-1 were treated with HMBA (1 mM) plus bryostatin-1 (20 nM) with or without ASA (2.5 mM), as described in the legend to panel A. ASA and SB202190 (50 μM) were used 2 h before bryostatin-1 treatment, where indicated. As a positive control for reactivation from latency, anti-CD3/CD28 Ab-conjugated Dynabeads were used (25 μl/1 × 10⁶ cells) to stimulate the cells and were given at the same time as bryostatin-1. The luciferase assay was performed upon lysis of cells using 100 μl of Bright-Glo substrate. Statistical analysis was made using one-way ANOVA (posttest, Newman-Keuls test) (*, P < 0.05; **, P < 0.01; n.s., not significant). (C) c-Jun expression correlates with the expression of HIV-1 Gag, used as a measurement of HIV-1 escape from latency obtained with HMBA plus bryostatin-1 treatment of the CD4⁺ T_CM cell latency model. WCEs (20 μg) derived from the same experimental points described in the legend to panel C were collected 4 h after bryostatin-1 treatment, separated by SDS-PAGE on a 10% polyacrylamide gel, and subjected to Western blotting analysis using specific Abs against c-Jun, HIV-1 Gag, and actin.