![Figure 1. Effect of ferroportin and ferroportin mutants on cell-surface transferrin receptor 1 and intracellular ferritin levels. (A) 293T cells at 30% to 50% confluence were transiently transfected with vectors encoding CD8, wtFPN–c-Myc, and the following c-Myc–tagged FPN mutants: C326Y, A77D, V162Δ, N144H, N144D, G490D, Y64N, Q248H, and L170F. After 2 days cells were double stained for CD8 or c-Myc tag and surface TfR1 and analyzed by 2-color flow cytometry. Transfected cells (> 70% of the population) expressing either CD8 or c-Myc were gated on and compared for their surface TfR1 levels. A comparison (top) is shown of TfR1 levels on cells positive for CD8 (gray filled histogram) versus untransfected cells (black line), demonstrating that expression of CD8 has no effect on TfR1. In all other panels, the gray-shaded histogram is TfR1 on CD8-transfected cells, and TfR1 on cells expressing wtFPN or mutant FPN is shown by a black line. Compared with CD8, TfR1 is increased on cells expressing wtFPN, indicating iron deficiency. C326Y, N144H, N144D, Q248H, and Y64N all increase expression of TfR1 in the same way as wild type, but A77D, V162Δ, and G490D do not increase TfR1 compared with CD8. L170F partially increases TfR1. The filled purple histogram represents the background fluorescence of cells stained with an isotype control antibody. (B) 293T cells were transfected as in panel A, but this time in the presence of 1 mg/mL Tf to increase background ferritin levels. After 2 days, cells were analyzed for total cell ferritin by ELISA. Cells were lysed in NP-40 at 107 cells/mL, doubling dilutions were made, and 20 μL was transferred to ELISA plate in triplicate. Graph shows mean total ferritin in ng/107 cells (± 95% CI [confidence interval]). Cells transfected with wtFPN have around 40 ng ferritin/107 cells, a 4-fold reduction compared with untransfected and control cells transfected with CD8. C326Y, N144H, N144D, Q248H, and Y64N all decrease ferritin levels in the same way as wild type, but A77D, V162Δ, L170F, and G490D do not significantly reduce ferritin. Asterisk indicates significance compared with untransfected or CD8 control (P < .001 by Student t test). Data shown is representative of 2 or more experiments for each mutant.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/105/10/10.1182_blood-2004-11-4502/6/zh80100578460001.jpeg?Expires=1724290132&Signature=w-WAwy86Z-AtjlRH2XowrlCHKoyrGTehJtBak-T~4DMSVYFhIdxmd4dDpyP1Du9H0DEMb2vzTubiPylfKkee-MWz8DKEXpvsLEbAnaubk5~8qRVGeTzg5967865lFMjm3eT1ax~6MeMbZBslt4vO89TWsR0Ll9SJ68arkPIoIekOTHDXeRBV-xZ860yxW4bmGFzW3TVIGsXdOcQ-8vK1uRvAQ0caRoBsH2CLCzLUwCEfA~h07wsw5KSji5ukYXDnKa~VIXeyt6or6Hdwo7fkJRYpVIWAazYOEOWk~lx9c6U4dwheTPGTV2iATiyjgFWpgP5mjbaIMGAsdY-QLPWwMg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Effect of ferroportin and ferroportin mutants on cell-surface transferrin receptor 1 and intracellular ferritin levels. (A) 293T cells at 30% to 50% confluence were transiently transfected with vectors encoding CD8, wtFPN–c-Myc, and the following c-Myc–tagged FPN mutants: C326Y, A77D, V162Δ, N144H, N144D, G490D, Y64N, Q248H, and L170F. After 2 days cells were double stained for CD8 or c-Myc tag and surface TfR1 and analyzed by 2-color flow cytometry. Transfected cells (> 70% of the population) expressing either CD8 or c-Myc were gated on and compared for their surface TfR1 levels. A comparison (top) is shown of TfR1 levels on cells positive for CD8 (gray filled histogram) versus untransfected cells (black line), demonstrating that expression of CD8 has no effect on TfR1. In all other panels, the gray-shaded histogram is TfR1 on CD8-transfected cells, and TfR1 on cells expressing wtFPN or mutant FPN is shown by a black line. Compared with CD8, TfR1 is increased on cells expressing wtFPN, indicating iron deficiency. C326Y, N144H, N144D, Q248H, and Y64N all increase expression of TfR1 in the same way as wild type, but A77D, V162Δ, and G490D do not increase TfR1 compared with CD8. L170F partially increases TfR1. The filled purple histogram represents the background fluorescence of cells stained with an isotype control antibody. (B) 293T cells were transfected as in panel A, but this time in the presence of 1 mg/mL Tf to increase background ferritin levels. After 2 days, cells were analyzed for total cell ferritin by ELISA. Cells were lysed in NP-40 at 107 cells/mL, doubling dilutions were made, and 20 μL was transferred to ELISA plate in triplicate. Graph shows mean total ferritin in ng/107 cells (± 95% CI [confidence interval]). Cells transfected with wtFPN have around 40 ng ferritin/107 cells, a 4-fold reduction compared with untransfected and control cells transfected with CD8. C326Y, N144H, N144D, Q248H, and Y64N all decrease ferritin levels in the same way as wild type, but A77D, V162Δ, L170F, and G490D do not significantly reduce ferritin. Asterisk indicates significance compared with untransfected or CD8 control (P < .001 by Student t test). Data shown is representative of 2 or more experiments for each mutant.
