FIG. 5.

DIO-1 upregulates and increases activation of caspases. (A) DIO-1 overexpression upregulates procaspase levels. FL5.12 cells were cultured in the presence of IL-3 (WT) or IL-3-starved for 24 h (WT-IL3). A plasmid vector (pcDNA3) or a DIO-1-expressing construct (DIO-1) were transiently transfected into FL5.12 cells and analyzed 24 h posttransfection. All cells were incubated with z-VAD-fmk (100 μM) to block proteolytic processing of the procaspase, facilitating accurate quantitation of protein levels. Procaspase 3 and 9 levels were analyzed in Western blots with anti-caspase 3 (upper panel) or anti-caspase 9 antibodies (middle panel). Loading was controlled with β-actin (lower panel). (B) Conditions as for panel A, but apoptosis was induced by IL-3 removal in pcDNA3-transiently transfected FL5.12 cells. Cells were cultured in the absence of z-VAD-fmk to measure caspase activity. Caspase 3-like, caspase 2, caspase 6, and caspase 9 activities were determined by fluorescence emission of the cleaved substrates (Materials and Methods). Data are given as the mean ± standard deviation for at least three independent experiments.