HT-29 cells were seeded at a density of 0.5 Â 10 6 cells per well in six-well plates and allowed to adhere overnight, then cotransfected with 3 mg of DRAK2 siRNA and 1 mg of pEGFP-N1 (as a marker to select transfected cells) DNA per well using Fugene 6.0 (Roche Diagnostics Corp.) with a 2 mg:1 ml ratio of RNA to transfection reagent. Transfections were performed in OptiMEM, 1 ml per well (final concentration of siRNA, approximately 200 nM). A negative control siRNA to a scrambled target in the DRAK2 gene (without sequence homology to any other known transcript as verified by BLAST analysis) was used in the mock transfection controls. Populations of both mock-and positively transfected cells were subsequently treated with either SC236 (5 mM), staurosporine (1 mM) or vehicle control for 24 h. Adherent and floating cells were then pelleted, washed and then re-suspended in 250 ml of PBS with 7-aminoactinomycin D (7-ADD; Molecular Probes) at a final concentration of 20 mg ml À1 and incubated on ice for 20 min in the dark. Samples were analysed by flow cytometry using an established method for assessment of cell viability in transfected cells selected according to GFP fluorescence (Vezina et al, 2001 ).