Nucleic Acids Research, 2011, Vol. 39, No. 12 5289 Figure 1. Splice-correction activity of PF14- SCO nanocomplexes in HeLa pLuc705 cell-line. HeLa pLuc705 cells (5 104) were seeded 24 h prior to experiments into 24-well plates. Cells were treated with PF14-SCO nanocomplexes at three different concentrations at MRs (3:1, 5:1, 7:1 and 10:1) for 4 h in serum-free medium followed by addition of serum to ﬁnal concentration of 10% and incubated additionally for 20 h (A), and at MRs (3:1, 5:1, 10:1 and 20:1) for 24 h in serum-containing medium (B). The controls used were: stearyl-TP10 (MR5, 200 nM), PF14 A (unstearylated PF14, Table 1) at MR5 and 200 nM dose, PF14 only (1 mM) and P14-unrelated ON (MR5, 200 nM). LF2000 was used according to the manufacturer’s protocol using the highest SCO concentration (200 nM). Cells were lysed in 0.1% Triton X-100 and luciferase activity was measured. Splice-correction assay results are presented as fold-increase over untreated cells (control). The values represent the mean of at least three experiments performed in duplicate (mean ± SEM, n = 3). Intergroup differences were considered signiﬁcant at *P < 0.05 using Student’s t-test. experiments were performed where PF14-Cy5-labeled SCO nanocomplexes together with labeled dextran were co-incubated with U2OS cells and extensive co- localization was observed (Figure 4C). Similar pattern was also seen in the A549 cell-line (Supplementary Figure S3). In addition, ﬂow-cytometry analysis was carried out to assess the percentage of cells being trans- fected. In keeping with the high splice-correction activity, we found that PF14 transfected nearly the entire cell popu- lation (Supplementary Figure S4). PF14 induces SCO-mediated splice-correction in a conﬂuence-independent and non-toxic manner It is a well-known fact that many commercial transfection reagents, such as LF2000, are strictly dependent on certain cell densities for optimal performance. To assess the effect of cell conﬂuency, transfections were carried out at differ- ent cell densities (2.5 104, 5 104, 10 104 and 20 104 cells/well), comparing PF14 with LF2000. As shown in Figure 5A, PF14-SCO complexes appeared less sensitive