Journal: PLOS Pathogens

Article Title: GS143, an inhibitor of E3 ligase β-TrCP, reverses HIV-1 latency without activating T cells via unconventional activation of NFκB

doi: 10.1371/journal.ppat.1013018

Figure Lengend Snippet: Abbreviations: gluc – Gaussia luciferase gene; T2A – T2A peptide promoting “ribosomal skipping”; EGFP – enhanced green fluorescent protein gene. (B) Resting CD4+ T cells infected with gGn-p6* were treated with indicated concentration of GS143, 10 nM Velcade, anti-CD3/CD28 beads plus 100 U/ml IL-2, 10 ng/ml phytohaemagglutinin (PHA) plus 100 U/ml IL-2, or 0.5 μM ionomycin plus 100 ng/ml phorbol 12-myristate 13-acetate (PMA) or with DMSO (NC negative control) in the presence of Raltegravir. GFP mean channel fluorescence from the infected cells was measured 48 hours later. Data from donor samples (n = 3 to 5) are presented as medians, interquartile ranges (IQR) and minimum and maximum with each donor represented by a dot are shown. Significance was determined by one-way ANOVA and Dunnett’s multiple comparisons comparing each treatment to untreated negative control (*p<0.05, **p<0.005 and ***p<0.0005). AU stands for arbitrary units. (C) HIV-1 mRNA expression in the resting CD4+ T cells (3~5 × 10 5 ) from healthy donors (n = 6) was measured by RT-qPCR following treatment with Chidamide (20 µM), JQ1 (1µM), Bryostatin (10 nM), GS143 (20 µM), PMA (100 ng/ml) + Ionomycin (0.5 µM) or DMSO (NC negative control) in the presence of Raltegravir. RNA measurements were normalized to the negative control. Each dot corresponds to a different donor and represents the average of two PCR reactions. Data are presented as medians, interquartile ranges (IQR) and minimum and maximum. Statistical significance was determined by non-parametric Wilcoxon matched-pairs signed rank test (two tailed). Statistically significant changes relative to NC were indicated as *P<0.05. In and , each individual is represented by specific symbol in different treatments so that the dose (where applicable) and treatment effect can be followed for an individual donor.

Article Snippet: The following drugs were used in cell treatment experiments: ionomycin, phorbol 12-myristate 13-acetate (PMA), Prostratin, Ingenol-3-angelate (I3A), N,N′-Hexamethylene bis(acetamide) (HMBA), TNFα (Sigma-Aldrich), anti-CD3/anti-CD28 coated magnetic beads (Thermo Fisher), recombinant human interleukin-2 (IL-2, PeproTech), Chidamide, pyroxamide (+)-JQ1 (Cayman Chemical), Bryostatin-1 (Calbiochem), Velcade (Selleck Chemical),GS143 (Tocris), Inhibitors BAY117085, LY2409881, and SC-514 (Selleckchem), Ethacrynic acid (Sigma).

Techniques: Luciferase, Infection, Concentration Assay, Negative Control, Fluorescence, Expressing, Quantitative RT-PCR, Two Tailed Test