Zfra antagonizes the apoptotic function of WOX1 and JNK1. (A) L929 cells were transfected with a cytotoxic dose of EGFP-WOX1 in the presence or absence of low doses of EGFP-Zfra by CaPO4 (n = 8). The cells were cultured 24 and 48 hrs and stained with crystal violet. Zfra blocked WOX1-mediated growth suppression and death in 24 hr (data not shown for 48 hr). Expression of EGFP-tagged Zfra and WOX1 is shown, as determined by fluorescence microscopy (400× magnification). (B) Similarly, L929 cells were electroporated with WOX1 and/or Zfra, cultured 48 hr, and then processed for DNA fragmentation. An apoptosis-inducing amount of WOX1 and a non-apoptosis-inducing dose of Zfra were used. In a positive control, cells were treated with staurosporine (stauro; 500 nM) for 16 hr. In negative controls, cells were electroporated with medium or GFP vector only. ep, electroporation. (C) L929 cells were electroporated with an apoptosis-inducing amount of WOX1 and/or Zfra, or JNK1 (or medium only), and cultured for 24 and 48 hrs, respectively. Apoptosis occurred in 24 hr, as evidenced by increased percentages of cells in the subG1 phase from cell cycle analysis (data not shown for 48 hr). WOX1, JNK1, or Zfra alone suppressed cell growth, as indicated by reduced cell populations at the G1. However, both Zfra and WOX1 or JNK1 could not increase apoptosis in a synergistic manner. X-axis: DNA content; Y-axis: Cell numbers.