FIG. 6.

Uncoupling protein 2 (UCP2) expression induces cell death. (A) Trypan Blue count. Viability of transfected cells: 3% in Lipofectamine and noncoding and vector-transfected cells, 11% in sense UCP2-transfected cells. (B) Live staining with Annexin-V in C-UCP2-transfected cells (left panel) and noncoding UCP2-transfected cells (right panel). Green is the green fluorescent protein (GFP) signal and red is Annexin-V. (C) Flow cytometry analysis with propidium iodide in C-UCP2-transfected cells (black) and noncoding UCP2-transfected cells (white). (D) Apoptosis-related gene expression analyzed by western blot from total cellular protein preparations (except for released cytochrome c, which was cytoplasmic protein preparation without mitochondria) of Hepa 1–6 cells 72 hr after transfection. (E and F) Rabbit polyclonal antibodies against Bcl-2 (sc-492; 1:1000 dilution) and Bax (sc-6236, 1:1000 dilution) were from Santa Cruz; rabbit polyclonal antibody against caspase 3 (1:1000 dilution) was from Cell Signaling; mouse monoclonal antibody against cytochrome c (1:300); and anti-bcl-x (1:500 dilution) was from BD Biosciences. Western blot samples from left to right: Transfections with C-UCP2, N-UCP2, noncoding UCP2, and vector alone, respectively. Color images available online at www.liebertpub.com/met