Fig. S1 DR4-selectivity and pro-apoptotic potential of N199R/K201H and G131R/D218H TRAIL mutants. (A) DR4-responsive ML-1 acute myelogenous leukaemia cells (B) and EM-2 chronic myelogenous leukaemia cells (C) as well as DR5-responding A2780 ovarian carcinoma cells (C) were treated with increasing doses of WT rhTRAIL, rhTRAIL-C3, G131R/D218H and N199R/K201H for 24 hrs. Induction of apoptosis was determined using annexin V assay and flow cytometric analysis. The graphs presented demonstrate the percentage of dead cells ± S.E.M. as determined from three independent experiments.

Fig. S2 Gel filtration elution profile of WT rhTRAIL (A) and rhTRAIL-C3 (B) from Hiload Superdex 75 16/60 column.

Fig. S3 Time versus response SPR sensorgrams. Receptor binding of WT rhTRAIL (A, C) and rhTRAIL-C3 (B, D) towards DR4-Ig (A, B) and DR5-Ig (C, D). Receptor chimeras were coated at a level of ~600--1000 RU. Purified WT rhTRAIL and rhTRAIL-C3 were injected in 3-fold at concentrations ranging from 250 to 2 nM at 70 μl/min. flow rate using HBS-P (Biacore) as running and sample buffer. Binding of ligands to the receptors was monitored in real time at 37°C.

Fig. S4 Receptor binding of WT rhTRAIL and rhTRAIL-C3 variant determined by SPR and competitive ELISA. Pre-steady state receptor binding curves of WT rhTRAIL and rhTRAIL-C3 to DR4-Ig (A), or to DR5-Ig (B) as determined by SPR. The response at each concentration was recorded 30 sec. after the end of the injections. Competitive ELISA assay of WT rhTRAIL and rhTRAIL-C3 for TRAIL receptors using immobilized DR4-Ig receptor and soluble DR4-Ig as competitor (C), soluble DR5-Ig as competitor (D), soluble DcR1-Ig as competitor (E) or soluble DcR2-Ig as competitor (F). Binding was calculated relative to the value measured in the presence of 0 ng/well of soluble receptor.

Fig. S5 Inhibition of NF-κB activity with the IKK inhibitor BMS-345541 (Calbiochem) enhanced the biological activity of both WT rhTRAIL and rhTRAIL-C3. HL-60 cells were treated with 100 ng/ml (A) or 10 ng/ml (B) of WT rhTRAIL or rhTRAIL-C3 in the presence or absence of the indicated concentrations of BMS-345541 for 12 hrs. Induction of cell death was determined by measuring the loss of mitochondrial membrane potential with TMRE. The graphs show average percentage of cells with low mitochondrial membrane potential (ΔΨm).

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