Fig. 1
Design, expression and specificity of sTRAIL specific variants. a Schematic drawing of sTRAIL constructs, all of which contain a heterologous signal peptide sequence from the human fibrillin-1 gene (hFIB) ligated to a Furin cleavage site (Furin CS), an Isoleucine Zipper (ILZ) domain and the soluble part of TRAIL (aa114-aa281). The expression was driven by a conventional CMV promoter/enhancer element (CMV). The mutations in sTRAILwt leading to the two sTRAILDR5 (TRAIL-R2 specific) and three sTRAILDR4 (TRAIL-R1 specific) variants are shown in the respective sTRAIL segments. b Results of ELISA analyses for TRAIL showing the levels of secreted sTRAILwt (yellow), sTRAILDR5–1 (dark green), sTRAILDR5–2 (light green), sTRAILDR4–1 (dark blue), sTRAILDR4–2 (light blue) and sTRAILDR4–3 (blue-grey) into the supernatant of HEK293 cells that were transfected with the described constructs. Results for cells transfected with an EGFP control expression construct (ctrl; grey) are also shown. c FACS histogram of HCT116 cells showing membrane expression levels of TRAIL-R1 (red) and TRAIL-R2 (blue). The FACS profile of the isotype control is shown as filled black. d Supernatants from HEK293 cells transfected with either an EGFP control expression plasmid (grey), sTRAILwt (yellow), sTRAILDR5–1 (dark green), sTRAILDR5–2 (light green), sTRAILDR4–1 (dark blue), sTRAILDR4–2 (light blue) or sTRAILDR4–3 (blue-grey) were normalised to 2 ng/ml TRAIL (the EGFP control was diluted 1:2 in fresh medium) and then applied to HCT116 (left), HCT.shDR4 (centre) and HCT.shDR5 cells (right), respectively, before apoptosis was measured 24 h later