Figure 2

Hepcidin reverses ferroportin-mediated inhibition of HIV-1 transcription. A, Ferroportin expression reduces ferritin level in 293T cells. 293T cells were transiently transfected with c-Myc-tagged WT ferroportin-expression vector (panel 2), or mutant c-Myc-tagged C326Y ferroportin -expression vector (panel 3). At 24 hrs posttransfection, the cells were incubated with 20 μM Ferric Ammonium Citrate for 16 hours and then additionally treated with 100 μM cycloheximide for 1 h followed by treatment with 0.3 μM hepcidin for 4 hrs. Ferritin concentration was analyzed by ELISA and normalized to total protein concentration that was determined by Bradford assay. B, Hepcidin reduced ferroportin expression. Protein samples prepared as in panel A were analyzed by SDS-PAGE and Western blotting using c-Myc antibody (upper panel), or tubulin antibodies as loading control (lower panel). C, Hepcidin restores HIV-1 transcription inhibited by ferroportin. 293T cells were transfected with vectors expressing CD4, WT ferroportin or mutant ferroportin C326Y. At 24 hrs posttransfection, the cells were re-transfected with HIV-1 LTR-lacZ and HIV-1 Tat expression vectors and immediately treated with indicated concentrations of hepcidin for 24 hrs. After the treatment, the cells were lyzed and β-galactosidase activity was determined using ONPG-based assay. Results are presented relatively to the control that was not treated with hepcidin. D, Hepcidin has no effect on transcription from CMV promoter. 293T cells were transfected with CMV-EGFP vector, treated with hepcidin at 24 hrs posttransfection and incubated another 24 hrs. The cells were lysed and EGFP fluorescence was measured on Luminescence spectrometer. Results are presented relatively to the control that was not treated with hepcidin.