Cells and media
Normal human dermal fibroblasts (NHDF) were obtained from ATCC, USA. DM1 fibroblasts (GM03132) were from Coriell Institute, USA. Southern blotting analysis showed that the expanded allele contained approx. 2250 CTG repeats in the DMPK gene. Standard medium: DMEM from Invitrogen™ with 10% fetal calf serum (Sigma-Aldrich), glutamine, streptomycin and penicillin. Low serum medium (HS): F12 medium from Invitrogen™ containing 3% horse serum, glutamine, streptomycin and penicillin.
Lentiviral production and transduction
The lentiviral vector encoding myoD was generated by replacing the puro gene in pCCL-WPS-PGK-puro-WHV  with myoD cDNA. For lentiviral production, 293T cells were seeded at 3 × 106 cells/p10 dish in standard medium, which was refreshed one hour prior to transfection. Cells were transfected by a CaPO4 co-precipitation method with 3.75 μg pMD.2G, 3 μg pRSV-Rev, 13 μg pMDGP-Lg/RRE and 13 μg of transfer vector (either pCCL-WPS-PGK-MyoD-WHV or pCCL-WPS-PGK-GFP-WHV). The medium was refreshed 24 hours post-transfection. One day later, supernatant containing the viral vector was filtered through a 0.45 μm pore filter, and polybrene added to a final concentration of 8 μg/ml. The medium was diluted 1:3 with standard medium and transferred to NHDF and DM1 fibroblasts. Medium was refreshed 24 hours after transduction.
RNA was isolated and cDNA was synthesized according to manufacturer's protocol (Sigma® and BioRad®, respectively). Before use, the cDNA was thawed and diluted appropriately; in this range of experiments a dilution of 1:4 was used. Furthermore, dilutions were made to set up a standard curve for the reactions. 2 μl of cDNA 1:4 dilution of each sample was added to 23 μl solution consisting of 12.5 μl TaqMan® universal PCR mastermix, 1.25 μl TaqMan mRNA specific primer set and 9.25 μl H2O. Reaction plates were analyzed by an ABI Prism® 7000 sequence detection system (Applied Biosystems). All samples were analyzed as triple determinations. The following TaqMan® assays were used: myogenin (MYOG, Hs01072232_m1, Applied Biosystems) and myoD (MYOD1, Hs00159528_m1, Applied Biosystems).
Human primary fibroblasts were grown in either standard medium or F12 with 3% horse serum (HS medium) at 37°C in 5% CO2. The cells were grown to appropriate confluence and fixed in 4% formaldehyde (Lilly's solution) for 10 minutes. After fixation, the cells were washed 3 times in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM NaH2PO4 (pH 7.2)). To lower the non-specific binding, the slide was incubated in 100 μl 1% BSA in PBS (blocking buffer) prior to antibody addition. Slides were washed in PBS and incubated with 100 μl primary antibody diluted (1:100) in blocking buffer (BSA-PBS) for 2 hours. 3× wash in PBS followed before 100 μl secondary antibody (1:400) diluted in blocking buffer, was added. The slide was incubated with secondary antibody for 1 hour before it was washed and 100 μl antifade, with or without DAPI (4',6-diamidino-2-phenylindole) was added. Primary antibodies were mouse monoclonal anti-myoD and anti-myogenin (both Santa Cruz Biotechnology). The secondary antibodies were Alexa Flour® 488 conjugated goat anti mouse (Invitrogen). All secondary antibodies were diluted 1:400 in 1% BSA PBS solution.
Fluorescence in situ hybridization (FISH)
FISH was performed according to protocol from SingerLab Online .
Probes were RP-HPLC purified and labeled with Cy3 (red) purchased from DNA Technology A/S, Denmark. The Cy3-labeled probe consisted of a (CAG)10-sequence with a fluorophore at the 5'-end . All reagents used in the above protocol were nuclease free and dissolved/diluted in nuclease free/DEPC-treated water or PBS unless otherwise stated. The numbers of RNA foci were counted microscopically and compared using Student's t-test.
Quantification of foci
The numbers of foci per cell were quantified by two different methods giving essentially similar results. The first method was based on direct counting, using a fluorescence microscope. Microscope fields were chosen randomly, using the DAPI filter, and dots were counted in all cells where the complete cell was present in the field. In another method images were acquired. This was done (in the Cy3 channel), using multidimensional acquisition, and images were stacked into a 2d-picture. The counting was performed by allowing the software to count the number of foci based on an arbitrary threshold value for minimum light intensity. Software used in the image acquisition was MetaMorph, and in the image processing, including counting, ImageJ was used.
Combined IF and FISH
Cells were rehydrated and permeabilized according to the FISH procedure described above. Next, cells were blocked in 1%BSA solution in a humidified hybridization chamber for 1 hour at room temperature. Afterwards, cells were transferred to a new hybridization chamber and incubated with primary antibodies for 1.5 hours at 37°C. Following this, cells were washed three times 5 minutes in PBS containing 5 mM MgCl2 at room temperature and thereafter incubated in secondary antibodies for 45 minutes at 37°C in a humidified hybridization chamber. Subsequently, cells were washed five times 5 minutes in PBS containing 5 mM MgCl2 at room temperature. Cells were fixed in 4% paraformaldehyde for 20 min at room temperature and the FISH procedure was performed as described above.
Transfection with RNA oligonucleotides
Oligonucleotides used in the present study comprised 21 nucleotides. They were chemically modified, as previously described  with a phosphorothioate backbone (PT) and methylated at 2'O (2'OMe). The antisense oligonucleotide had the sequence (CAG)7, targeting the pathogenic repeat. The control oligonucleotide had the following sequence: GUAGCGACUAAACACAUCAAG.
Cells were transferred to glass cover slips in a 12-well plate with a confluency of 1.5 × 105 cells/well. The following day, cells were transfected with the oligonucleotide, using polyethyleneimine (PEI) aided by hyaluronic acid (HA) as transfection reagent (Sigma-Aldrich). Transfection was performed as previously described .
Oligonucleotides (600 μg/ml), hyaluronic acid (HA) (Sigma-Aldrich; 3.5 mg/ml) and polyethyleneimine (PEI) (Sigma-Aldrich; 937.5 μg/ml) were mixed in a 1:2:1 ratio (by volume). This suspension was diluted in an equal volume of doubly concentrated PBS. The DNA/HA/PEI/PBS solution was incubated for 30 minutes at room temperature. Immediately before adding the complex to the cells, the medium was refreshed (ordinary DMEM) and the complex was added to the cells. Cells were incubated for 4 hours at 37°C with the transfection complex and the medium was subsequently replaced with fresh DMEM. Cells were fixed for FISH analysis 24 hours post-transfection.