Fig. 3. BRD4 inhibition alleviates HIV-1 latency.

(A) U1 cells were stably transduced with the indicated shRNAs, then HIV-1 gag mRNA levels were measured by qPCR and normalized to the shFLuc control.

(B) Western blot of cells in (A).

(C) A transcriptional elongation assay was performed as above using cells in (A). The level of transcripts was normalized to the shFLuc control. Note log scale on the Y axis.

(D) A transcriptional elongation assay was performed as above using U1 cells treated with either JQ1 (500 nM) or DMSO for 1 hr. The level of transcripts was normalized to the values of the DMSO-treated controls. Note log scale on the Y axis.

(e) U1 cells were treated with either JQ1 (500 nM) or Prostratin (1 uM) alone, or the combination. After 72 hr cDNA was prepared and levels of gag mRNA determined.

(f) J-Lat A2 cells were treated with JQ1 (500 nM) or Prostratin (1 μM) for 72h. Cells were then assessed for GFP expression and viability. For the cell viability assays the relative luminescence units (RLU) of DMSO treated cells was set at 100%.

(g) The indicated J-Lat cell lines were treated with JQ1 (500 nM), PMA (200 nM), or the both for 72 hr. GFP positive cells were assessed using flow cytometry, and the percentage of positive cell was calculated.

(h) The indicated J-Lat cell lines were treated with the noted compounds (JQ1 (500 nM), TNF-α (10 ng/ml) alone or in combination. At 72 hr the percentage of GFP positive cells was determined by flow cytometry.

Values represent the mean +/− S.E.M., N > 3 throughout. * P < 0.05, ** P < 0.01. Results were analyzed by unpaired t tests. See Fig. S3.