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. 2017 Jul 20;13(7):e1006460. doi: 10.1371/journal.ppat.1006460

Fig 8. Inhibition of CD40L selectively affects WT HIV-1.

Fig 8

(A) IPA diagram highlighting genes in the CD40L pathway that are susceptible to down-regulation by digoxin. HIV-1 WT preferentially integrates within or near these genes. Continuous lines indicate direct and experimentally validated interactions between genes; dashed lines indicate experimentally validated, indirect interactions. (B-D) Purified memory CD4+ T-cells were stimulated via CD3/CD28 Abs, cultured for 3 days and exposed to the indicated concentrations of digoxin for 24h. Cells were labeled with anti-CD40L FITC-conjugated antibodies, stained for cell viability and analyzed by flow cytometry. (B) Gating strategy to select single, live CD4+ T-cells. (C) Representative plot showing the percentage of memory CD4+ T-cells expressing CD40L on their surface in the presence of the indicated concentrations of digoxin (y-axis). (D) The percentage of CD40L positive cells and the MFI were quantified by flow cytometry. Bar graphs represent mean ± SD of 3 donors. Friedman test p-values are indicated on the graphs: *, p<0.05; ** p<0.01. (E) Jurkat cells were infected with HIV-1 LAIΔenv GFP WT or N74D at MOIs of 0.05–0.2 in the presence of the indicated concentrations of the anti-CD40L neutralizing antibody and analysed by flow cytometry 48h after infection to measure the percentage of GFP+ cells. Dot plots showing the results of five independent experiments. Unpaired, two-tailed t-test p-values are indicated on the graphs: *, p<0.05; **, p<0.01.