Figure 4

Hepcidin mediates degradation of ferroportin and partially restores inhibition of HIV-1 transcription. A and B, Hepcidin mediates degradation of WT but not C326Y ferroportin. 293T cells were transfected with vectors expressing WT ferroportin-EGFP, mutant C326Y ferroportin-EGFP or non-relevant control NIPP1 pA-RATA-EGFP. Transfected cell were treated with FAC for 24 hours and then with 0.5 μM hepcidin (A) or with indicated concentrations of hepcidin (B) for 18 hours. EGFP fluorescence was measured in intact cells using FACS (A) or in cell lysates using Luminescence spectrometer (B). C, Ferroportin reduces inhibition of HIV-1 transcription from HIV-1 proviral DNA by WT ferroportin-EGFP but not mutant C326Y ferroportin-EGFP. 293T cells were transfected with vectors expressing EGFP-fused wild WT ferroportin or mutant ferroportin C326Y and also co-transfected with HIV-1 Luc genomic clone. The cells were treated with FAC for 24 hours and treated with indicated concentrations of hepcidin. After 24 hours of treatment, the cells were lyzed and luciferase activity was determined. EGFP fluorescence was also determined and used to normalize the results.