Figure 2.

Effect of unmodified and stearylated cell-penetrating peptides (CPPs) on plasmid transfections compared to the Lipofectamine 2000 (LF2000). (a) To evaluate the ability of unmodified CPPs to mediate plasmid transfections, 4 × 10⁴ Chinese hamster ovary cells were seeded 24 hours before experiment in 24-well plates. Cells were treated with complexes at different charge ratios (CRs), from CR1 to CR4 (in this case CR3 is shown), using 0.5 µg of plasmid per well, for 4 hours in serum-free media (alternatively with the addition of 100 µmol/l chloroquine (CQ)) followed by replacement to 10% serum containing medium and incubated additionally for 20 hours. Cells were washed with HEPES-buffered Krebs Ringer buffer and lysed in 0.1% Triton X-100, luciferase activity was measured and normalized against the protein content in each well. (b) Transfection comparison of stearyl-transportan 10 (TP10) and stearyl-Arg9 at CR3, carried out as described above. (c) Efficiency of stearyl-TP10 compared to LF2000. Stearyl-TP10 was formulated at different CRs as described above and LF2000 was used according to the manufacturer's protocol. (d) Decrease in LF2000-mediated luciferase plasmid transfections as a result of decreased LF2000 amounts compared to the standard protocol. Uptake of the fluorescenyl-labeled plasmid in complex with either (e) TP10 or stearyl-TP10 in U2OS cells in Opti-MEM or (f) serum containing media. Treatments were carried out as described above, however, cells were lysed for 1 hour and fluorescence was measured in black 96-well plate at 490/518 nm on a fluorometer. Fluorescence signal (RFU) from untreated cells was subtracted from the signals of treated cells. The values represent the mean of at least three independent experiments performed in duplicate (mean ± SEM). (b) ***P < 0.001, analysis of variance Dunnett's multiple comparison test.