C282Y HFE induces cytochrome c release and caspase-3 activation. HEK293 cells (1 × 106) were transfected with EV, WT HFE, C282Y HFE expression plasmids or untransfected cells treated with 2.5 μM thapsigargin, and cytosolic extracts were prepared 24 h posttransfection. Ten-microgram of protein was assayed for cytochrome c release (A), and caspase-3 activation (B) by western blotting (n = 3). C282Y HFE (M) induced cytochrome c release and processing of procaspase-3 compared to EV, WT HFE or 2.5 μM thapsigargin treated cells. (C) To confirm equal loading blots were stripped and probed for β-Actin. (D) Measurement of caspase-3 activity in the lysates of HEK293 cells (1 × 106) transfected with control (empty vector), WT HFE or C282Y HFE expression vectors, and positive control of 2.5 μM thapsigargin was used. C282Y HFE significantly increased caspase-3 activity (*, p = 0.0363) (n = 3).