Fig 7. Synergistic inducibility of HIV-1 LTR promoter by PKC agonist+BETi/HMBA combinatory treatments depends on NF-κB.

Panel A. Jurkat cells were transiently transfected with the episomal plasmid containing the luciferase reporter gene driven either by the wild-type HIV LTR promoter (LTRwt-luc) or by the LTR promoter mutated in the two NF-κB binding sites (LTR-NFκBmut-luc). Twenty-four hours later, cells were mock-treated, treated with JQ1 (0.5μM), I-BET (0.5μM), I-BET151 (0.5μM), HMBA (5mM), bryostatin-1 (10nM) and prostratin (2.5μM) alone or in combination. Luciferase activities in cell extracts were measured 24 hours after drug treatments and reported as fold increases over the activity observed in mock-treated conditions (transfection of the reporter plasmid without drug treatment) and arbitrarily set at values of 1. An experiment performed in triplicates representative of two independent experiments is shown. Panel B. Nuclear extracts were prepared from Jurkat cells which were mock-treated, treated with bryostatin-1 (10nM), JQ1 (0.5μM) or with bryostatin-1+JQ1 for different time periods. An oligonucleotide corresponding to the HIV-1 LTR NF-κB sites was used as probe in EMSAs. As control for equal loading, the bottom panel shows comparability of the various nuclear extracts assessed by EMSA with an Oct-1 consensus probe.