Fig. 2: Potency of HIV latency reversal by VEH < VEH | SIV ECs and THC | SIV ECs.

A) Representative western blot image of intracellular expression of HIV antigens – p24 and p17 in U1 cells, with host (U1 cells) GAPDH used as loading control. B) The levels of HIV gag p24 expression (shown in panel A) analyzed by densitometry (ImageJ) and presented as fold change (treatment/PBS). C) The level of extracellular HIV RT released by EC-treated U1 cells into the culture supernatants after 4 days of treatment. D-G) Clarified U1 cell supernatants collected on day 4 of treatment were added to the indicator cells – TZM-GFP cells and cultured for (D, E) one day or (F, G) two days. D, F) Representative microscopic images of HIV infection (GFP expression, green) after 1 and 2 days of infection respectively. E, G) Quantification of HIV infection (GFP expression, green) after 1 and 2 days of infection respectively. The numbers below the bars indicate fold change (treatment/PBS). “No virus” panel shows the level of autofluorescence detected by the instrument. H-K) The same clarified U1 cell supernatants collected on day 4 of treatment were added to CEMx174 cells and cultured for (H, I) one day or (J, K) four days. H, J) Representative microscopic images of cell morphology. The number of syncytia and large cells were quantified on I) day 1 and K) day 4. Red, blue, and green arrows denote syncytia, large cells, and filopodia-like protrusions respectively. L, M) Clarified supernatants collected on day 4 from EC-treated U937 cells were added to CEMx174 cells and cultured for 4 days. L) Representative microscopic images of cell morphology. M) The number of syncytia and large cells were quantified. N, O) CEMx174 cells were treated with VEH, VEH | SIV, THC | SIV ECs for 4 days. No virus = cells treated with PBS but not media from EC-treated U1 cells. No CM = cells treated with regular media but not conditioned media. N) Microscopic image of cell morphology. M) The number of syncytia and large cells were quantified. P) Schematic of HIV transfer assay from CEMx174 cells infected with EC-induced HIV cocultured with uninfected indicator TZM-GFP cells. Q) Representative microscopic images of HIV transfer assay from CEMx174 (red) to TZM-GFP cells. Green and orange (green + red) indicate infection. R) The level of infection (GFP expression). The numbers below the bars indicate fold change (treatment/PBS). All experiments were repeated three times. Statistical differences were assessed by ordinary one-way ANOVA with Tukey’s correction and by Binary Student’s t tests (Welch’s correction). **** p < 0.001, *** p < 0.005, ** p < 0.01, * p < 0.05, and ns = non-significant. Scale bars (horizontal orange lines) = 200 µm.