Figure 3.

DIO-1 induces apoptosis in vitro. The DIO-1 ORF was cloned into the pcDNA3 mammalian expression vector (Invitrogen). Both empty vector and the DIO-1 construct were transiently transfected by electroporation into A20 and Ba/F3 cell lines. After 48-hr expression, the cells were permeabilized and stained with propidium iodide and cell cycle analyzed by FACS. FL5.12 wild-type and stably transfected hBcl-2 cells were transiently transfected as before. DIO-1NLSpcDNA3 encodes a mutant protein lacking amino acids 162–192, which is therefore unable to translocate to the nucleus. Where indicated, the general caspase inhibitor Z-VAD-fmk (50 μM final concentration; Bachem) was added immediately after transfection.