Figure 2

C282Y HFE activates EOR. (A) NF-κB-luciferase measurement. HEK 293 cells (1 × 10⁶) were transfected with pcDNA3.1 (empty vector, EV), WT HFE, or C282Y HFE (M) expression plasmids and an NF-κB-luciferase reporter gene vector. As positive control, five hours post transfection cells transfected with NF-κB-luciferase reporter gene vector were stimulated with 2.5 μM thapsigargin for 24 h. Cells were incubated for 24 h in serum-complete medium. Luciferase production in cell lysates was measured by luminometery. Levels are expressed as light units (LU) per microgram of total protein (***, p = 0.0009) compared to WT HFE. Assays were performed in triplicate and are representative of at least three separate experiments. (B) Degradation of IκB-α by C282Y HFE. HEK 293 cells were transfected with pcDNA3.1 (empty vector, EV), WT HFE, or C282Y HFE expression plasmids, and cytosolic extracts were prepared 24 h post-transfection. Ten-microgram of protein was assayed for IκB-α degradation by Western blotting (n = 3). (D) HEK 293 cells (1 × 10⁶) were transfected with pcDNA3.1 (empty vector, EV), WT HFE, or C282Y HFE expression plasmids and an IL-8-luciferase reporter gene vector. As positive control, five hours post transfection cells transfected with IL-8-luciferase reporter gene vector were stimulated with 2.5 μM thapsigargin for 24 h. Cells were incubated for 24 h in serum-complete medium. Luciferase production in cell lysates was measured by luminometry. Levels are expressed as light units (LU) per microgram of total protein of total protein (**, p = 0.0031) compared to WT HFE. Assays were performed in triplicate and are representative of at least three separate experiments.