For cell transfection, the RUNX1 isoforms p48, p38a, p30 and p24 as well as the CD56 isoforms CD56120kDa and CD56140kDa were cloned into the mammalian expression MIDGE-vector pMCV1.2. 2x10^6 K562, HL-60 and U937 cells were transfected with 1-3 µg of plasmid DNA using solution V and program T-019 (HL-60) and T-003 (K562). For controls, equal amounts of pmaxGFP expressing plasmid were used to nucleofect cells under identical conditions. Transfection efficieny was determined as the proportion of GFP positive cells assessed with a fluorescent microscope and ranged between 60% (HL-60) and 85% (K562).
For RNAi, K562 and HL-60 cells were nucleofected with 50 nM RUNX1-exon 3, -exon 5a and -exon 5.4 as well as AlexaFluor488 labelled negative control siRNA duplexes. Transfection efficiency (~98%) was determined by fluorescence microscopy.