Figure 5: AZT induces ASK1 and promotes the antiviral activity of A3G.

(a,b) PBMC or H9 cells were treated with the indicated agents (0–10 μM) for 24 h. The expression of ASK1 was confirmed by western blotting. APV, amprenavir; ATV, atazanavir; AZT, zidovudine; DRV, darunavir; IDV, indinavir; LPV, lopinavir; NFV, nelfinavir; NVP, nevirapine; SQV, saquinavir; 3TC, lamivudine. (c) H9 cells were transfected with control siRNA or ASK1-targeted siRNA for 48 h before infection of HIV-1. At 2 days after infection, cells were washed and additionally cultured for 24 h in the presence or absence of AZT (10 μM). Cells and supernatants were then harvested and analysed by western blotting against the indicated antibodies. We used AZT-resistant virus to avoid the effects of AZT carry-over from the culture supernatant of producer cells during the reverse transcription step in target cells. (d) TZM-bl reporter cells were infected with harvested and normalized virus to measure viral infectivity (n=3, mean±s.d.). Full images for all western blots analysis are shown in Supplementary Fig. 6.