FIG 5.

c-Jun is a crucial factor mediating HIV-1 reactivation from latency in J-Lat 10.6 2NΔ4 cells. (A and B) Inhibition of c-Jun expression by stable genetic knockdown determines a significant reduction of the percentage of J-Lat 10.6 2NΔ4 cells reactivating from latency upon combined stimulation with bryostatin-1 and ionomycin. For each experimental point, 0.2 × 10⁶ 2NΔ4 cells expressing control shRNA (sh ctr) and c-Jun-specific shRNA (sh c-Jun) were treated with ionomycin (A) at a concentration of 0.5 μg/ml or with HMBA (B) at a concentration of 1 mM for 72 h before FACS analysis and with bryostatin-1 (A and B) at a concentration of 20 nM for 24 h before FACS analysis. The graphs show the level of reactivation of virus transcription, measured by the percentage of cells expressing GFP. Data from 4 separate experiments are presented as box plots indicating the median and the interquartile range. Statistical analysis was made using the two-tailed paired t test (*, P < 0.05; **, P < 0.01). (C) Stable genetic knockdown of c-Jun in J-Lat 10.6 2NΔ4 cells results in the inhibition of HIV-1 reactivation from latency. A total of 1 × 10⁶ cells stably expressing control or c-Jun-specific shRNA were treated with a combination of ionomycin (0.5 μg/ml) or HMBA (1 mM) plus bryostatin-1 (20 nM) with HMBA or ionomycin, both of which were administered 48 h before bryostatin-1 treatment. WCEs (50 μg) were collected 4 h and 18 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, GFP, and actin. Different DMSO controls were used to normalize different volume dilutions of each inhibitor compounds. (A to C) ctr, control; I, ionomycin; H, HMBA; B, bryostatin-1.