Zfra induces apoptotic cell death. (A) Transiently overexpressed Zfra induced death of human ovarian ME180, embryonic kidney HEK-293, neuroblastoma SK-N-SH, breast MDA-MB-231 and MCF7 cells, Molt4 T lymphocytes and murine L929 fibroblasts, but not human prostate DU145 and mink lung epithelial Mv1Lu cells. These cells were transfected with an EGFP (vector) or EGFP-Zfra expression vector (150 ng/well in 96-well microtiter plates) or medium (control) by CaPO4, followed by culturing for 48 hr and then staining with crystal violet for determining the extent of death (n = 8; mean ± standard deviation; p < 0.001, EGFP vector-transfected cells versus EGFP-Zfra-transfected cells except DU145 and Mv1Lu; Student's t tests). Molt4 T cells were transfected with the DNA constructs by electroporation. Data shown in the buffer and vector (EGFP) controls are from testing L929 cells. Similar data were obtained using other cells. The numbers of cells with positive EGFP or EGFP-Zfra protein expression were ~60–75%, as determined by fluorescence microscopy. EGFP-Zfra was present ubiquitously in cell compartments in majority of the tested cells (see representative L929 cells; 200× magnification). Interestingly, EGFP-Zfra was mainly present in the cytoplasm of MDA-MB-231 cells (400× magnification). (B) In adherence- or anchorage-independent growth assay on agarose, Zfra was shown to block colony formation of L929 cells, as opposed to empty vector controls (n = 3, p < 0.001; Student's t test). Live colonies were stained with a soluble tetrazolium-based MTS proliferation reagent. (C) Molt4 T cells were electroporated with the EGFP-Zfra expression vector, followed by culturing for 48 hr and analyzing the extent of apoptosis using Annexin V assay.