Figure 1.

Expression of functional rFasL and enhanced green fluorescent protein in HEK-293T cells. HEK cells were transfected with the control plasmids CE3 [14] and pCX-eGFP or the rFasL-expressing plasmids pCE-rFasL-IRES-EGFP and pCX-rFasL-IRES-EGFP. (A): Reverse transcription-polymerase chain reaction experiments demonstrated transcription of the rFasL gene in EF- and XF- but not in the E_gfp- and X_gfp control-transfected HEK cells. (B): Western blot assays using the rabbit anti-mouse FasL (N-20) antibody from Santa Cruz Biotechnology showed that naïve and X_gfp-transfected HEK cells had no expression of a FasL protein, whereas a FasL-specific protein was detected in rat thymus and rFasL-transfected HEK cells. The latter cells also showed several other forms of the rFasL protein. (C–E): The functionality of FasL expression was determined by coculture experiments with Jurkat cells. (C): As control for apoptosis induction, Jurkat cells were incubated with 50 ng/ml sFasL for 24–48 hours and analyzed by fluorescent activated cell sorting (FACS) using propidium iodide (PI) (supplemental online Fig. S2). There was a reduction of cells when sFasL was added to the cultures, and this effect could be blocked by Fas/Fc (100 ng/ml). (D): Control HEK, HEK.X_gfp, and HEK.XF cells were cocultured with Jurkat cells for 48 hours and analyzed by FACS in the presence of PI (see supplemental online Fig. S2 for details). The data were plotted as ratios of Jurkat cells over HEK cells. (E): When compared with cocultures with naïve or green fluorescent protein-expressing HEK cells, there was a reduction of cells in the Jurkat cell gate in the presence of rFasL-transfected HEK cells, and this effect could be blocked in the presence of Fas/Fc. Abbreviations: EF, pCE-rFasL-IRES-EGFP; E_gfp, CE3; HEK, human embryonic kidney; rFasL, rat Fas ligand; sFasL, soluble Fas ligand; XF, pCX-rFasL-IRES-EGFP; X_gfp, pCX-eGFP.