Figure 3

HIV activation by TNFα and TSA alone or in combination with Aza-CdR in four J-Lat cell lines. A. GFP positive cell quantification by FACS analysis at 48 h post-induction of J-Lat 6.3, 8.4, 9.2 and 10.6 cell lines treated with TNFα or Aza-CdR alone or in combination. 20.000 events from the live population defined by forward versus side scatter gating were analyzed. B. Proviral activation as determined by percent GFP positive cells for the different treatments and treatment combinations in the four J-Lat cells (J-Lat 6.3, 8.4, 9.2 and 10.6 indicated on each panel). C. p24 antigen production for all treatments and treatment combinations at 48 h post-induction. D. Cell viability of the different treatments and treatment combinations determined with an MTS assay (Promega, Madison, WI) at 48 h. Cells were cultured in RPMI with 10% FBS. Two million cells were seeded and treated with the different compounds for 48 h. The compound order of addition was determined as described in Fig. 1: agents were added at time 0 h, with no further additional steps. After 48 h, cells were washed twice with chilled 1× PBS and fixed in 2% formaldehyde as in the FACS analysis described in the Fig. 1 legend. At the same time, supernatants from the different treatment conditions were stored at -80°C until further use. p24 antigen was determined using ELISA (Perkin Elmer, Waltham, MA) from the stored supernatants. Final activator compound concentrations were, for Aza-CdR (2.5 μM), TNFα (10 ng/ml) and TSA (1.5 μM). Results are the mean ± standard deviation (SD). Statistical analysis (Student's t-test) was performed using the STATA software package (StataCorp LP, College Station, TX), * p ≤ 0.05.