Figure 4

Activation of the ATF-6, CHOP by C282Y HFE. HEK 293 cells were cotransfected with pcDNA3.1 (empty vector, EV), WT HFE, or C282Y HFE (M) expression plasmids and (A) ATF6-promoter luciferase reporter gene vector (ATF6-luc) (B) CHOP-promoter luciferase reporter gene vector (CHOP-luc), with or without pZeoSv2⁺ (data not shown). As positive control or treatment, five hours post transfection cells were incubated with 2.5 μM thapsigargin or 300 μM TUDCA 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. C282Y HFE activated ATF-6 and CHOP-luc expression (**, p = 0.0039 and **, p = 0.0011 respectively) compared to WT HFE. Treatment with TUDCA significantly decreased ATF6-luc and CHOP-luc expression (#, p = 0.0175 and ##, p = 0.0067 respectively) compared to WT HFE. Z A1AT transfection significantly increased ATF6-luc and CHOP-luc expression (*, p = 0.02 and **, p = 0.0059) compared to WT HFE. Assays were performed in triplicate and are representative of at least three separate experiments.