4% paraformaldehyde (PFA) and stained for MyoD, Pax7, and GFP, as previously described[44]. Primary Myoblast Isolation and Culture Primary myoblasts were isolated from hind limb skeletal muscle. Muscles were minced and digested in type I collagenase and dispase B mixture (Roche Applied Science). Cells were then filtered from debri, centrifuged, and cultured in growth media (F- 10 Ham’s medium supplemented with 20% fetal bovine serum, 4 ng/mL basic fibroblast growth factor, and 1% penicillin- streptomycin) on collagen-coated dishes at 37uC, 5% CO2. Differentiation media consisted of DMEM supplemented with 5% horse serum, 1% penicillin-streptomycin Myoblast cultures testing the presence of recombinant Dlk1 protein were maintained on a bed of 1 mg/mL BD Matrigel (Basement Membrane Matrix; BD Biosciences, San Jose, CA, USA). Experimental cultures also contained 500 ng/mL of recombinant Dlk1 protein (Dlk1(mouse): Fc(human), cat#ALX- 201-416-C010; Alexis Biochemicals-Enzo Life Sciences Interna- tional, Inc. Plymouth Meeting, PA, USA) in the Matrigel bed. Cells were maintained in the same culture media conditions as listed above for proliferation and differentiation. Transfection of C2C12 Cells and primary myoblasts C2C12 cells and primary myoblasts were transfected using the NeonTM Transfection System (Invitrogen, Inc) according to the manufacturer’s recommended protocol. Dlk1 or GFP-containing plasmids were transfected at a rate of 2 mg of plasmid DNA in 26105 cells. C2C12 cells were then plated in 6-well plates and grown in DMEM with 10% FBS, 1% penicillin/streptomycin. Primary myoblasts were grown under identical conditions as described above in the Primary myoblast isolation and culture section. RNA Isolation and Quantitative PCR Analysis Tissue samples for RNA were excised after euthanization and either stored in RNAlater (Ambion, Woodlands, TX) for later processing or homogenized immediately for RNA purification using the Qiagen RNeasy Fibrous Tissue Mini Kit (Qiagen, Inc., Valencia, CA). The on-column DNase treatment included in the kit was used to remove any trace amounts of genomic DNA. Purified RNA samples were then quantified by fluorometry (Quant-iT Ribogreen RNA Quantitation Kit, Invitrogen-Molec- ular Probes, Eugene, OR). Equal amounts of RNA were reverse transcribed using random hexamer primers and M-MLV reverse transcriptase (Invitrogen, Inc., Carlsbad, CA). Quantitative PCR was performed using the Roche Lightcycler 480 system with Roche SYBR Green Master Mix reagents (Roche Applied Science, Indianapolis, IN). Samples were assayed in duplicate with 80 ng of cDNA per 10 uL reaction. Ribosomal protein large protein 38 (Rplp38) was used as the housekeeping gene for all gene expression studies. Primer sequences and PCR conditions are listed in Supplemental Table S2. Fold expression values relative to the wild-type samples within each tissue were calculated using the 22DDCT method. Statistical significance was determined by ANOVA using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC, USA). Western Blots and Electrophoretic Mobility Shift Assay (EMSA) Levels of different proteins in skeletal muscle were determined by performing Western immunoblotting as described [88,89]. Briefly, tissues were washed with phosphate-buffered saline (PBS) and homogenized in western blot lysis buffer (50 mM Tris-Cl Role of Dlk1 in Myogenesis and Satellite Cells [pH 8.0], 200 mM NaCl, 50 mM NaF, 1 mM dithiotheritol (DTT), 1 mM sodium orthovanadate, 0.3% IGEPAL, and protease inhibitors). Approximately 100 mg of protein were resolved on each lane on 10–12% SDS-PAGE, electrotransferred onto nitrocellulose membrane, and probed with specific antibodies (Supplemental Table S1) and detected by chemiluminescence. The bands were quantified using ImageQuant TL software (GE Healthcare). NF-kB activation in skeletal muscle was analyzed by EMSA as previously described [88] with some modifications. In brief, TA muscles isolated from mice were immediately frozen in liquid nitrogen and suspended at 1 mg muscle weight per 18 ml of low salt lysis buffer (10 mM HEPES [pH 7.9], 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2.0 mg/ml leupeptin, 2.0 mg/ml aprotinin, 0.5 mg/ml benzamidine) followed by mechanical grinding using mortar and pastle. Cells in the lysis buffer were allowed to swell on ice for 10 min followed immediately by three cycles of freeze-thaw lysis. The tubes containing the lysed muscle cells were then vortexed vigorously for 10 s, and the lysate was centrifuged for 30 s at 14,000 rpm. The supernatant (cytoplasmic extracts) was removed and saved at 270uC for further biochemical analysis. The nuclear pellet was resuspended in 4 ml of ice-cold high-salt nuclear extraction buffer (20 mM HEPES [pH 7.9], 420 mM NaCl, 1 mM EDTA, 1 mM EGTA, 150 mM MgCl2, 25% glycerol, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2.0 mg/ml leupeptin, 2.0 mg/ml aprotinin, 0.5 mg/ml benzamidine) per mg of original muscle weight and was incubated on ice for 30 min with intermittent vortexing. Samples were centrifuged for 5 min at 4uC, and the supernatant (nuclear extract) was either used immediately or stored at 270uC. The protein content was measured by the method of BioRad (Hercules, CA) protein assay reagent. EMSAs were performed by incubating 20 mg of nuclear extract with 16 fmol of the 32P-end-labeled NF-kB consensus oligonucleotide 59-AGT TGA GGG GAC TTT CCC AGG C-39 (Promega, Madison, WI) for 15 min at 37uC. The incubation mixture included 2–3 mg of poly dI.dC in a binding buffer (25 mM HEPES [pH 7.9], 0.5 mM EDTA, 0.5 mM dithiothreitol, 1% Nonidet P-40, 5% glycerol, 50 mM NaCl). The DNA-protein complex thus formed was separated from free oligonucleotide on 7.5% native polyacrylamide gel, using buffer containing 50 mM Tris, 200 mM glycine (pH 8.5), and 1 mM EDTA. The gel was dried, and the radioactive bands were visualized and quantitated by a PhosphorImager (GE Healthcare), using ImageQuant software. Supporting Information Figure S1 Localization and expression of Dlk1 in skeletal muscles. A: Lack of Dlk1 expression in quiescent Pax7+ satellite cells in a cross section of a resting muscle. B: Co- localization of Dlk1 with interstitial CD31+ endothelial lineage cells. C: Up-regulation of Dlk1 expression in newly regenerated myofibers (small caliber fibers with central nuclei) but not in Pax7+ satellite cells in a regenerating muscle 5 days after cardiotoxin treatment. D: Relative Dlk1 mRNA expression in whole muscles, single myofibers isolated from EDL muscle and cultured myoblasts (n = 18, 4, 2 respectively; * indicates significant difference compared to whole muscle). (TIF) Table S1 List of antibodies used in this study. (DOC) PLoS ONE | www.plosone.org 10 November 2010 | Volume 5 | Issue 11 | e15055