Expression plasmids for pcDNA3-MEF2D, pCMV β-galactosidase [28, 29], and reporter gene constructs for 3TP-lux , MCK-Luc , and MEF2-Luc  have been previously described. KLF6 reporter constructs pRMO6 and pROM6 ΔMEF2 were generously provided by Dr. Nicolas P. Koritschoner (Faculty of Bioquimica y Ciencias Biologicas, Universidad Nacional del Litoral, Santa Fe, Argentina).
Anti-MEF2A rabbit polyclonal, anti-Myosin heavy chain mouse monoclonal and anti-Myogenin mouse monoclonal antibodies were produced with the assistance of the York University (Toronto, Ontario, Canada) Animal Care Facility. Anti-MEF2D (1:1000; BD Biosciences, Mississauga, Ontario, Canada), Smad3, phospho-Smad3 and phospho-ERK1/2 (1:1000; Cell Signaling, Toronto, Ontario, Canada), and KLF6, actin, and ERK1/2 (1:1000; Santa Cruz, Santa Cruz, CA95060, US) were used for immunoblotting experiments. Immunoglobulin G (IgG) was also purchased from Santa Cruz Biotechnologies.
Cell culture, transfections and drug treatments
C2C12 cells were maintained in DMEM supplemented with 10% fetal bovine serum (HyClone, Rockford, IL61101, US), 1% L-glutamine and 1% penicillin-streptomycin. Cells were maintained in a humidified, 37°C incubator with a 5% CO2 atmosphere. For transfections, cells were seeded on pre-gelatin-coated plates 1 day prior to transfection and were transfected according to the standard calcium phosphate method previously described by Perry et al., 2001. A mixture of 50 μl 2.5 M CaCl2 per 25 μg DNA with an equal volume of 2× HeBS (2.8 M NaCl, 15 mM Na2HPO4, 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH = 7.15) was used, and the cells were incubated overnight followed by washing and addition of fresh media. Drug treatments were used at the following concentrations: 2 ng/ml TGFβ, 5 μM Sis3 and 10 μM U0126 as indicated.
siRNA gene silencing
siRNA targeting KLF6, MEF2D and non-specific scramble RNA were purchased from Sigma. Transient transfections were performed using TurboFect Transfection Reagent (R0531, Fermentas) according to the manufacturer’s instructions. Turbofect (Fermentas): a 1:2 mixture ratio of DNA to turbofect reagent (including 4 ng/ml siRNA) in 200 μl serum-free DMEM was prepared for 19-h incubation.
C2C12 cells were treated as previously described by Salma and McDermott, 2012 , and incubated overnight with at 4°C with primary MEF2D and KLF6 antibodies (1:100) diluted in 1.5% goat serum. Cells were washed three times with PBS for 10 minutes and incubated with the appropriate tetramethyl rhodamine iso-thiocyanate (TRITC)/fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (1:500) in 1.5% goat serum (PBS) for 2 h at room temperature (RT) following 4’,6-diaminidino-2-phenylindole (DAPI) staining for 15 minutes at RT. Cells were washed three times with PBS and cover slips were mounted with DAKO mounting media (Dako) on glass slides. The fluorescence images were captured using Fluoview 300 (Olympus).
Protein extractions, immunoblotting and reporter gene assays
Cells were harvested using an NP-40 lysis buffer (0.5% NP-40, 50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 10 mM sodium pyrophosphate, 1 mM ethylenediaminetetraacetic acid (EDTA) (pH 8.0), 0.1 M NaF) containing 10 μg/ml leupetin and aprotinin, 5 μg/ml pepstatin A, 0.2 mM phenylmethylsulfonyl fluoride and 0.5 mM sodium orthovanadate. Protein concentrations were determined using the Bradford method (Bio-Rad) with BSA as a standard. We used 20 μg of total protein extracts for immunoblotting, diluted in sample buffer containing 5% β-mercaptoethanol, and boiled. Transcriptional assays were done using Luciferase reporter plasmids. The cells were harvested for these assays using 20 mM Tris, (pH 7.4) and 0.1% Triton-X 100, and the values obtained were normalized to β-galactosidase activity expressed from a constitutive SV40-driven expression vector and represented as relative light units (RLU), or in some cases, corrected Luciferase values for control, reporter alone transfections were arbitrarily set to 1.0, and fold activation values were calculated. Bars represent the mean (n = 3) and error bars represent the standard error of the mean (n = 3).
Protein extracts were prepared as described above. Immunoprecipitation was performed using the ExactaCruz kit (Santa Cruz Biotechnology), as per manufacturer’s instructions. Precipitated proteins were separated by SDS PAGE and immunoblotting of proteins was performed as described above.
Chromatin immunoprecipitation (ChIP)
ChIP experiments followed the guidelines set by EZ ChIP™ (Upsate) with minor modifications. Approximately 1× 107 C2C12 cells were fixed with 1% formaldehyde (Sigma) for 15 minutes at 37°C. Fixing was quenched by Glycine (Bioshop, Burlington, ON Canada) at a final concentration of 0.125 M. Cells were collected in PBS containing phenylmethylsulfonyl fluoride (PMSF) (Sigma) and protease inhibitor cocktail (Roche, Laval, Quebec, Canada). Cells were collected at 5000 rpm for 5 minutes at 4°C. Cells were lysed using Wash Buffer I (10 mM HEPES pH 6.5, 0.5 M ethylene glycol tetraacetic acid (EGTA), 10 mM EDTA, 0.25% Triton X-100, protease inhibitor cocktail, PMSF) for 5 minutes on ice. Nuclei were collected and resuspended in Wash Buffer II (10 mM HEPES pH 6.5, 0.5 mM EGTA, 1 mM EDTA, 200 mM NaCl, protease inhibitor cocktail, PMSF) for 10 minutes on ice. Nuclei were again collected and then treated with nuclear lysis buffer (50 mM Tris–HCl pH 8.1, 10 mM EDTA, 1% SDS). Chromatin was sheared using a Misonix sonicator to produce 500 bp fragments. Crosslinked sheared chromatin was collected following a 15-minute spin at maximum speed. Twenty percent of total chromatin was set aside as input. Sheared crosslinked chromatin was diluted 1:10 with immunoprecipitation (IP) dilution buffer (0.01% SDS, 1.1% Triton-X 100, 1.2 mM EDTA, 16.7 mM Tris–HCl pH 8.1, 167 mM NaCl) and incubated with antibody overnight at 4°C with rocking. Protein G Dynabeads (Invitrogen) were blocked with 20 μg salmon sperm DNA in IP dilution buffer (15 μl of beads + 135 μl IP dilution buffer + 20 μg salmon sperm DNA per IP) overnight at 4°C with rocking. We incubated 152 μl of pre-blocked beads with the IP reaction at 4°C for 1 h. Dynabead-bound antibody-chromatin complexes were washed using IP Wash Buffer I (20 mM Tris pH 8.1, 2 mM EDTA, 150 mM NaCl, 1% Triton-X 100, 0.1% SDS) and II (20 mM Tris pH 8.1, 2 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.1% SDS), each incubated for 10 minutes at 4°C, and followed with two washes in Tris-EDTA (TE) buffer at 4°C. Protein-DNA complexes were freed from Dynabeads through the addition of elution buffer (0.1 M NaHCO3, 1% SDS) for 30 minutes at RT. To separate protein from DNA, samples were treated with 12 μl of 5 M NaCl (BioShop) at 65°C for 4 h or overnight. Protein was further degraded by the addition of Proteinase K (Sigma), EDTA, Tris pH 6.5 for 1 h at 45°C. DNA samples were then purified using a PCR clean up kit (Qiagen, Mississauga, ON, Canada).
ChIP-qPCR analysis of the KLF6 promoter was done using BioRad Sybr Green as per the user manual with a final primer concentration of 0.5 μM. The antibody used in ChIP was 5 μg αMEF2 (sc-313X; Santa Cruz Biotechnology, Inc.). The equivalent amount of rabbit IgG (12–370, Millipore) was used as a control in each ChIP. Sequences of the primers flanking the ME2 site on the KLF6 promoter were: 5’-CTGCAACGTTGGGCTGTA-3’ and 5’-TTGGAAAGACGTCTCACAGG-3’. Each sample was run in triplicate and then analyzed using percent input or fold enrichment.