Cell Culture
The mouse pre-myoblast cell line C2C12, was obtained from European Cell Culture ECCAC. C2C12 cells were maintained in Dulbeco’s Modified Eagle´s Medium (DMEM) with High glucose (Thermo) containing 10% of fetal bovine serum (Gibco) and antibiotics (100U/mL Penicillin and 100µg/mL of Streptomycin) and L-Glutamine (Thermo) at 37°C
in a humidified atmosphere of 5% CO2. Cells (3,5 x 105) were plated into 100-mm culture
dishes with regular growth medium previously described. At 24 h after plating, regular growth medium was replaced by 0,25% albumin as described [13] and maintained in culture medium depleted of serum for another 24 h.
RNA extraction, and quantitative real-time PCR analysis.
Total RNA was isolated from C2C12 cells in the presence or absence of BMP-2 (Thermo Fisher, PHC7141) and in presence or absence of nicotinamide (Sigma-Aldrich, N0636). Cells were washed with PBS and lyzed with TRIzol reagent (Invitrogen), and using the DirectZol RNA mini prep kit (Zymo research, #R2050). After RNA purification, the integrity was assessed by agarose electrophoresis and the quantification was estimated with the Qubit 2.0 instrument (Life Technologies). 2µg of total RNA was used for reverse transcription using M-MLV (Promega) and the product was then used for real-time PCR using the Maxima® SYBR Green/Rox qPCR Master mix 2X (Fermentas Life Sciences Cat. #K0221). PCRs were performed in triplicate for each cDNA the results were averaged. The relative mRNA levels were normalized to the reference Mouse Snrpd3 gene. The reaction was carried out in a Stratagene Mx3000 (Agilent Technologies) thermocycler. PCR primers were designed with Primer-BLAST program [14] using different exon sequences of the corresponding genes (Table 1).
Western Blot analyses
C2C12 cells were exposed to BMP-2 (500 ng/mL) and/or nicotinamide (20 mM) for 0, 0.5, 2 and 4 hours. Then total proteins lysate were extracted for later analyses. Halt Protease and Phosphatase inhibitor cocktail (Thermo Scientific #78440) was used to prevent degradation and dephosphorylation of proteins. Whole cell lysates were separated by SDS-PAGE electrophoresis using NuPAGE 4-12% Bis-Tris precast gel (Invitrogen) and the Bolt Mini Gel Tank System (Life Technologies) according to the instructions of the manufacturer. For inmunodetection pSmad 1/5/8 rabbit monoclonal antibody (Cell signaling, Danvers, MA, USA cat# 13820), GAPDH mouse monoclonal antibody (Santa Cruz Biotechnology sc-32233) were used. Anti- rabbit and Anti-mouse IgG conjugated with HRP (Sigma-Aldrich) was used as the secondary antibody respectively. Inmunoreactivities were detected by Pierce ECL Western Blotting Substrate (Thermo scientific).
Cell culture staining
Cells were seeded into a 24-well plate and cultured for 24 and 48 hrs in the same conditions described previously. Cultures were fixed with 4% Paraformaldehyde (Sigma-Aldrich) for 30 min and stained with Alkaline Phosphatase (Fast Blue RR salt, Sigma-Aldrich) according to the manufacturer´s instructions.
Alkaline Phosphatase activity colorimetric assay
Cells were seeded in 24-well plates at a density of 4,000 cells/well and grown in presence or absence of nicotinamide or BMP-2 for 24 and 48 h. The ALP activity was measured by colorimetric assay kit (K412-500, Biovision, USA). Briefly, cells were lysed in the ALP assay buffer, the supernatant was collected and incubated with p-nitrophenol phosphate at
25°C for 1 hour. After adding the Stop solution, the absorbance was measured at 405 nm by using Victor 1420 Multilabel counter (Perkin Elmer, Wellesley, MA). The protein content was measured in the cell lysate using the Qubit 2.0 fluorometer. Specific ALP activity was normalized using the total protein concentration.
Inmunofluorescent Analysis
Cells were seeded on a glass coverslides in 24-well plate and incubated in presence or absence of BMP-2 and nicotinamide for 0, 0.5, 2, and 4 hours. The cells were washed three times with PBS and fixed with 4% Paraformaldehyde (Sigma-Aldrich) for 30 min and then incubated with 0,5% Tritón X-100 (Sigma-Aldrich, X100-500ML) in PBS for 10 min. After washing 3 times with PBS, the coverslides were incubated in 1% bovine serum albumin (BSA) with 0,1% Tween 20 (Sigma-Aldrich) for 1 hour. Then incubated with primary antibodies and TRITC-conjugated goat anti-rabbit IgG or FITC-conjugated anti-mouse IgG secondary antibodies (Sigma-Aldrich). Images were acquired by the Epifluorescence microscope Nikon Eclipse E600.