One glo system

The recently reported TZA was used to measure the HIV-1 reservoir size as previously described (39 (link)). Resting CD4⁺ T cells isolated from HIV-1–infected, aviremic subjects were treated with LRAs (ingenol or ingenol + JQ1) or PLK1 inhibitors (SBE 13 HCl or UMB-158), alone or in combination, for 48 hours at 37°C. Cells were stimulated with human anti-CD3/CD28 Dynabeads (Gibco) for 4 days in RPMI complete medium containing efavirenz (300 nM). Cells were washed thoroughly with the medium, counted, serially diluted (fourfold), and cocultured with fresh TZM-bl cells seeded on 96-well plates (four replicates for each dilution). Uninfected resting CD4⁺ T cells from healthy donors were treated similarly in parallel as controls. At 48 hours after co-culturing, TZM-bl cells were harvested for luciferase activity assay (One-Glo System, Promega). Chemiluminescence was determined by using the Cytation 5 multimode reader. Signal from the well containing cells from HIV-1–positive subjects was counted as positive if it exceeded the mean ± 2 SD of the output obtained from the control well (HIV-1–negative subjects). The IUPM representing HIV-1 reservoir size was determined by applying the maximum likelihood estimate via an online tool available at http://silicianolab.johnshopkins.edu/, which was developed by Rosenbloom et al. (58 (link)).

Luciferase assays were carried out as described elsewhere (Uhlenhaut et al. 2013 (link)). In short, candidate enhancers were amplified from mouse genomic DNA using oligos that added 5′ XhoI and 3′ HindIII or BglII restriction sites. Oligo sequences are reported in the Supplemental Information. Cis-regulatory elements were cloned into pGL4.23 (Promega). After transfection, CV-1 cells were treated overnight with 1 µM Dex or ethanol, and reporter activity was determined with Promega's ONE-Glo system.