Fig. 2

Enhanced reactivation potential of romidepsin by ALKi-3 in lymphoid J-Lat 9.2 and promonocytic U1 in vitro latency cellular models. (A-B, F-G) J-Lat 9.2 and U1 cells were either DMSO-treated, treated with increasing doses of ALKi-3 [25-50-100-200 µM] alone or in combination with sub-optimal dose of romidepsin (17.5 nM, and 5 nM for J-Lat 9.2 and U1 cells, respectively). At 24 h post-treatment (A, F) viral reactivation was assessed by measuring the concentration of genomic viral RNA copies/ml in culture supernatant using RT-qPCR. (B, G) Cells metabolic activity was measured by using XTT assay. Results obtained with the mock-treated cells were arbitrary set at a value of 100%. (C-E, H-I) Cells were treated with either DMSO (control), ALKi-3 alone (100 µM), or in a combination with suboptimal dose of romidepsin (17.5 nM, and 5 nM for J-Lat 9.2 and U1 cells, respectively). Viral reactivation was assessed 24 h post-treatment. (C) J-Lat 9.2 cells were subjected to flow cytometry analysis to quantify the percentages of GFP⁺ cells. (D, H) Viral production was estimated by measuring CA-p24 antigen in culture supernatant. (E, I) Total RNA was extracted that was subsequently subjected to quantification by RT-qPCR for TAR- and RRE-containing HIV-1 RNAs. Values were normalized using gapdh primers and were presented as relative fold changes to the values measured in romidepsin + DMSO-treated cells which were arbitrarily set at a value of 1. (A-I) Means and standard errors of the means from three biological repetitions in duplicates are represented. Statistical analysis was performed using a paired Student’s t-test with p-values indicating the significance level: p ≤ 0.05 (*), p ≤ 0.002 (**), p ≤ 0.0002 (***), and p ≤ 0.0001 (****)