Figure 6

Taurosodeoxycholic acid inhibits C282Y HFE induced apoptotic responses. (A) HEK 293 cells (1 × 10⁶) were transfected with pcDNA3.1 (empty vector, EV), WT HFE, or C282Y HFE (M) expression plasmids and an bcl-2-luciferase reporter gene vector. As positive control, five hours post transfection cells 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. C282Y HFE decreased bcl-2-luc expression (**, p = 0.004) compared to WT HFE. Treatment with TUDCA significantly increased bcl-2-luc expression (#, p = 0.0238) compared to WT HFE. Z A1AT transfection significantly decreased bcl-2-luc expression **, p = 0.0029 compared to WT HFE transfected with M A1AT. Assays were performed in triplicate and are representative of at least three separate experiments. (B) HEK293 cells (1 × 10⁶) were transfected with C282Y HFE (M) with or without Z A1AT expression plasmids. Transfected cells were untreated or treated with 300 μM TUDCA, and cytosolic extracts were prepared 24 h posttransfection. (B) Ten-microgram of protein was assayed for cytochrome c release, and (C) caspase-3 activation by western blotting (n = 3). Incubation with TUDCA markedly inhibited cytochrome c release and processing of procaspse-3. (D) Blots were stripped and probed with β-Actin to confirm equal loading. (E) Measurement of caspase-3 activity in the lysates of HEK293 cells (1 × 10⁶) transfected with C282Y HFE (M) and either Z A1AT HFE or M A1AT expression plasmids. Transfected cells were untreated or treated with 300 μM TUDCA. Positive control of 2.5 μM thapsigargin used. Z A1AT increased C282Y HFE (M) caspase-3 activity compared to M A1AT (*, p = 0.0379) and treatment with 300 μM TUDCA significantly decreased C282Y HFE (M) induced caspase-3 activity compared to untreated (#, p = 0.0214).