5286 Nucleic Acids Research, 2011, Vol. 39, No. 12 Table 1. Sequences of PF14 and PS-20-O-methyl oligoribonucleotides Splice-correction assay Name PF14 PF14 A SCO for HeLa pLuc705 cells SCO for mdx mouse myotubes Sequence Stearyl- AGYLLGKLLOOLAAAALOOLL-NH2 AGYLLGKLLOOLAAAALOOLL-NH2 50-CCU CUU ACC UCA GUU ACA 50-GGCCAAACCUCGGCUUACCU coupling proceeded for 10 min. The crude ON was puriﬁed by anion-exchange chromatography and desalting fol- lowed by anion-exchange high-performance liquid chro- matography (HPLC) and mass analysis as previously described (22). The molarity of ONs was determined by measurements of optical density at 260 nm. The PS-20-O- Me RNA ON for mdx mouse myotubes (Table 1) with sodium counterions was obtained from RiboTask (23). PF14 (Table 1) was synthesized (SYRO multiple peptide synthesizer, MultiSynTech, Germany) on Fmoc-Rink- amide-4-methylbenzhydrylamine resin (0.67 mmol/g, IRIS Biotech, Germany) using standard Fmoc solid-phase peptide synthesis (24). Stearic acid (Sigma-Aldrich) was coupled to the N-terminus by using TBTU/HOBt [2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetraﬂuoroborate/1-hydroxybenzotriazole] (IRIS Biotech, Germany) activation in dimethylformamide/dichloro- methane. The peptide was cleaved using 95% TFA/2.5% water/2.5% triisopropylsilane for 3 h and precipitated in diethylether. The obtained crude peptide was dried in vacuum overnight. The peptide was puriﬁed by HPLC on a Discovery C-18 Supelco column (Sigma-Aldrich, Sweden) using a gradient of acetonitrile/ water containing 0.1% TFA. The identity of the puriﬁed product was veriﬁed by analytical HPLC and by Perkin Elmer prOTOFTM 2000 matrix-assisted laser desorption ioniza- tion time-of-ﬂight mass spectrometer (Perkin Elmer, Sweden). The mass-spectrum was acquired in positive ion reﬂector mode using a-cyano-4-hydroxycinnamic acid as a matrix (Sigma-Aldrich) (10 mg/ml, 7:3 acetoni- trile: water, 0.1% TFA). The molarity of the peptide was determined based on dilutions of accurately weighed substances. Cell culture HeLa pLuc705 cells, kindly provided by Prof. R. Kole, and U2OS cells were grown at 37 C, 5% CO2 in Dulbecco’s Modiﬁed Eagle’s Medium (DMEM) with glutamax supplemented with 0.1 mM non-essential amino acids, 10% fetal bovine serum (FBS), 200 U/ml penicillin and 200 mg/ml streptomycin (Invitrogen, Sweden). Mdx mouse myotubes were obtained from con- ﬂuent H2K mdx cells seeded in gelatin-coated 24-well plates following 2 days of serum deprivation (DMEM with 5% horse serum) (25). More information regarding cell culture and myotubes development can be found in Supplementary Figure S1. HeLa pLuc705 cells (5 104) were seeded 24 h prior to ex- periments into 24-well plates. PS-20-OMe SCOs were mixed with PF14 at different molar ratios (MRs) in MQ-water in 10% of the ﬁnal treatment volume (i.e. 50 ml). Complexes were formed for 1 h at room tempera- ture and meanwhile the cell medium was replaced in the wells with fresh serum-free or serum-containing DMEM media (450 ml). Thereafter, complexes were added to each well. When using LF2000, Oligofectamine, LipofectamineTM RNAiMAX (Invitrogen, Sweden) and jetPEI (Polyplus transfection, In Vitro, Sweden), the complexes were prepared according to the manufacturer’s protocol. Cells were treated with PF14-SCO nano- complexes at different MRs (3:1, 5:1, 7:1, 10:1, 20:1) for 4 h in serum-free medium (or in serum-containing media) followed by addition of serum to a ﬁnal concentration of 10% (only to cells grown in serum-free medium) and incubated for additional 20 h. Thereafter, the cells were lysed using 100 ml 0.1% Triton X-100 in HEPES-buffered Krebs Ringer (HKR) buffer for 30 min at room temperature. Luciferase activity was measured using Promega’s luciferase assay system on GLOMAXTM 96 microplate luminometer (Promega, Sweden) and normalized to protein content determined using the Lowry method (BioRad, USA). In experiments with chloroquine, after complex formation and prior to treatment of cells, chloroquine (ﬁnal concentration 100 mM) was added to the transfection mixture in order to promote endosomal escape. Four hours after addition of the nanocomplexes and chloroquine to cells, cell medium was replaced with fresh medium in order to avoid the toxic effects of chloroquine. In experiments as- sessing the impact of temperature on splice correction, cells were kept for 30 min at 37 or 4 C prior to addition of PF14-SCO complexes. Cells were treated for 1 h, washed twice with PBS and cultivated back at 37 C for additionally 20 h. For experiments with mdx mouse myotubes, PF14 nanocomplexes at different MRs were prepared as mentioned above and incubated with myotubes for 4 h in 0.5 ml OptiMEM or in DMEM/10% FBS. ONs complexed with Lipofectamine 2000 in OptiMEM according to the manufacturer’s protocol, were also incubated with myotubes in OptiMEM as a control. The media in all cases was replaced by 1 ml of DMEM/5% horse serum media for further incubation. Reverse-transcription PCR For the HeLa pLuc705 cell-line, cells were trypsinized and total RNA was isolated from the cell pellets using the RNeasy plus kit (QIAGEN, Sweden). The quality of RNA was veriﬁed by agarose gel electrophoresis. Three nanogram of RNA were used in each reverse-transcription PCR (RT–PCR) reaction in which the total volume was 20 ml using the ONE STEP RT–PCR kit (QIAGEN). The primers had the following sequences: Fwd-50-TTGATAT GTGGATTTCGAGTCGTC-30; Rev-50-TGTCAATCAG AGTGCTTTTGGCG-30. The program for the RT–PCR was as follows: 55 C, 35 min, then 95 C, 15 min, for reverse transcription step directly followed by the PCR