SPI-2 expression inhibits Fas-mediated DNA fragmentation and caspase 3 activation. (A) Immunoblot analysis of SPI-2 expression in stably transfected Jurkat cells. Jurkat cells transfected with SPI-2 BMGneo [JSPI-2(4) and JSPI-2(5)] (lanes 1 and 2) express SPI-2, whereas cells transfected with the empty vector (Jneo) (lane 3) do not. (B) Jurkat cells were treated with 250 ng of anti-Fas antibody per ml for 8 h, and DNA fragmentation was monitored by TUNEL assay as described in Materials and Methods. Panels: a, untreated Jneo cells; b, Jneo cells treated with anti-Fas; c, Jneo cells treated with anti-Fas in the presence of 100 μM zIETD-fmk; d, untreated JSPI-2 (clone 4); e, JSPI-2 (clone 4) treated with anti-Fas; f, untreated JSPI-2 (clone 5); g, JSPI-2 (clone 5) treated with anti-Fas. Representative data from three experiments are shown. (C) Caspase 3 activation in untreated Jneo cells (lane 1), Jneo cells treated with anti-Fas antibody for 8 h (lane 2), untreated JSPI-2 (clone 4) cells (lane 3), JSPI-2 (clone 4) cells treated with anti-Fas (lane 4), untreated JSPI-2 (clone 5) cells (lane 5), and JSPI-2 (clone 5) cells treated with anti-Fas (lane 6) was monitored by Western blotting.