Ethical procedures
All animal experiments were performed in compliance of Regulation № 20/01.11.2012 on the minimum requirements for protection and welfare of experimental animals and the requirements for the sites for their use, breeding and / or delivery, issued by the Ministry of Agriculture and Food of Republic of Bulgaria.
Mouse tissue samples collection
Five male white laboratory mice, 6-8 weeks old, were humanly euthanized. Tissue specimens were excised from the femoral and gluteal muscles and fixed with freshly prepared methacarn fixative [19], or stored at -80°C for further studies. Specimens from lungs, spleen, brain, liver, intestine, colon and kidneys were archived in low temperatures, too. After processing, the fixed specimens were embedded in paraffin.
Cell cultures
C2C12 mouse myoblast cell line (ATCC® CRL-1772™) was purchased from LGC Standards USA (Manchester, NH, USA). The cells were cultured for 48h in Dulbecco's Modified Eagle's Medium (DMEM, Sigma-Aldrich, St. Louis, MO, USA) with a high glucose content of 4.0 g / L supplemented with 10% fetal bovine serum (FBS, Gibco-Thermo Fisher Scientific, Waltham, MA, USA), penicillin 100 IU / ml and streptomycin 100 μg / ml, (AppliChem, Darmstadt, Germany) into plastic tissue culture flasks (Orange Scientific, Braine-l`Alleu, Belgium) or onto 12 mm oval glass cover slips (Glaswarenfabrik Karl Hecht GmbH, Sondheim, Germany), until 90% confluence of the monolayer was achieved. Further differentiation into myotubes was induced by changing the growth medium to differentiation medium – Dulbecco's Modified Eagle's Medium, supplemented with 2% horse serum (Sigma-Aldrich). The cells were dissociated by 0.05% solution of trypsin (Gibco-Thermo Fisher Scientific) with 0.025% ethylenediaminetetraacetic acid (AppliChem) and counted with an automatic cell counter (CountessTM, InvitrogenTM, ThermoFisher Scientific). Samples with approximate concentration of 5x106 cells/ml were stored at -80°C for further molecular and proteomic studies. The myotube layers onto the cover slips (Glaswarenfabrik Karl Hecht GmbH, Sondheim, Germany) were submitted for lectin cytochemistry.
Lectin histo- and cytochemistry
Cover slips with myotube cultures and skeletal muscle tissue sections were treated with biotinylated lectins – Maackia amurensis lectin (MAL, Vector Laboratories, Burlingame, CA, USA), specific for α-2,3-sialic acids [20] and Sambucus nigra agglutinin (SNA, Vector Laboratories), specific for α-2,6-bound sialic acids [21]. The tissue sections were first rehydrated, and the myotubes were treated with 0.3% Triton in buffer. The further steps were performed in dark. All samples were incubated for 30 min with 1 µg/mL methanol solution of 4`6-diamidino-2-phenylindole (DAPI, AppliChem, Darmstadt, Germany), then with SNA or MAL (1 µg/mL) for 60 min, and finally with Streptavidin-FITC (1:100, Sigma-Aldrich) for 30 min. Control samples were treated with buffer instead of lectins. The samples were mounted in Vectashield mounting medium (Vector Laboratories) and observed with light microscope Leica DM 5000B (Leica Camera AG, Wetzlar, Germany) under UV, blue, green and UV/violet fluorescent filters. The obtained parallel images of each sample were merged using ImageJ Software [NIH, USA, 22].
Gene expression analyses
The experiments described in this section were designed to evaluate the expression of mRNA of sialyltransferases ST3Gal1, 2, 3, 4 and 6, ST6Gal1 and 2, and ST6GalNAc1, 2, 3 and 4 in mouse skeletal muscle tissue samples and mouse C2C12 myotube cell cultures. The levels of expressions were estimated via normalization versus the expressions of peptidyl prolyl isomerase A (PPIA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as reference genes. All primers were designed using the NCBI Blast Tool [23] in a way to span at least one intron sequence. The full names of investigated genes, the primer sequences and the size of the amplified products are available as a supplementary file. The oligonucleotides were purchased from HVD Biotech Vertriebs (Vienna, Austria). The substrate specificities of all sialyltransferases analyzed in this study are shown in Table 1.
Five skeletal muscle tissue samples with approximate weight of 30 mg each and five aliquots of C2C12 myotube cell cultures from different passages with approximate concentration of 5x106 cells/ml were homogenized using Tissue Ruptur II homogenizer (Qiagen, Hilden, Germany) on ice. Total RNA was isolated and purified by GeneMatrix Universal RNA Purification Kit (EurX®, Gdansk, Poland), strictly following the corresponding protocols recommended by the producer. The yield and purity of the collected RNA were measured using S-300 Spectrophotometer (Boeco, Hamburg, Germany).
Approximately 2 µg total RNA from each sample were used for first strand cDNA synthesis. The RT Master Mix contained 5x Reaction Buffer, 20 U RiboLock Rnase Inhibitor, 1 mM dNTPs, 100 pmol Random hexamer primers, 200 U RevertAid Reverse Transcriptase (all of them Fermentas of Thermo Fisher Scientific) and DEPC-treated water (Sigma-Aldrich). The reaction mixture was first incubated at room temperature for 10 min, then at 42°C for 1h, and the reaction was terminated at 70°C for 10 min. The generated cDNA was quantified and the samples were stored at -80°C.
Real-time PCR was designed on approximately 700 ng cDNA as a template in 20 µl total volume of reaction using SG qPCR Master Mix (2x), 0.25 U uracyl-N-glycolase (UNG), nuclease free water (all from EurX) and 0.2 µM R- and F-primers specific for amplification of fragments of GAPDH, PPIA, ST3Gal1, 2, 3, 4 and 6, ST6Gal 1 and 2, and ST6GalNAc1, 2, 3 and 4. Three real-time PCR reactions/sample in duplicate were performed for amplification of each fragment of interest using 36-well RotorGene™ 6000 Real-time Analyzer (Corbett Life Science-Qiagen).
The data were analyzed using Rotor Gene Q Series Software (Qiagen) and the relative quantification of the STR expressions was calculated by the ΔΔCt method [26] versus PPIA and GAPDH as reference genes. After each run, a High Resolution Melting Curve Analysis (HRM) was performed to verify the specificity of the amplified products, which were visualized on 2.5% agarose gel supplemented with Simply Safe nucleic acid stain (EurX) versus 100-1000 bp DNA Ladder (EurX) and the gels were photographed with a gel documentation system Vision (Scie-Plas Ltd, Cambridge, UK).
Statistical analysis of the gene expression quantification
Statistical analysis of the data was performed using GraphPad Prism 5.03 software (San Diego, CA, USA). One Way Anova analysis with test of Bonferroni was computed to detect statistically significant differences between the Ct values of the qPCR products, and the results were interpreted as follows: P < 0.001 = highly significant, P < 0.01 = very significant, P < 0.05 = significant.
SDS-PAGE, lectin- and western blotting
Skeletal muscle tissue samples with an approximate weight of 30 mg each and aliquots of C2C12 myotube cell cultures from different passages with an approximate concentration of 5x106 cells/ml were homogenized in 0.6M Tris buffer, containing 150 mM NaCl, 5 mM EDTA and 1% CHAPS (all purchased from Sigma-Aldrich), supplemented with Proteinase inhibitor cocktail, set 3 (Sigma-Aldrich), using Tissue Ruptur II homogenizer (Qiagen) on ice, and then centrifuged at 21000 x g, for 1 h at 4°C. The supernatants were used for methanol/chloroform protein precipitation as described by Fic et al. [27]. The protein pellet was reconstituted in 6 M Urea buffer, containing 1.5 M Thiourea, 3% CHAPS, and 66 mM DTT (all purchased from Sigma-Aldrich), and stored at -20°C. The protein content was measured by the method of Bradford [28] on spectrophotometer S-300 (Boeco).
Approximately 30 µg from each sample were mixed with 4xLoading buffer (EurX), samples were heated at 98°C for 10 min and were then loaded on 10% polyacrylamide gel. SDS-PAGE was performed under reducing conditions as described by Laemmly [29].
Gels were then stained with colloidal coomassie brilliant blue [30], or were forwarded to western blotting on 0.45 µm nitrocellulose membranes (Sigma-Aldrich), as described by Towbin et al. [31]. The membranes designated for lectin-affino blots were blocked with 5% non-fat dry milk (Sigma-Aldrich) for 1 h, then incubated with biotinylated SNA (Vector Laboratories) or MAL (Vector Laboratories) (1 µg/mL) for 1 h, and finally treated with streptavidin horseradish peroxidase (HRP, Vector Laboratories) for 30 min at room T°C. The membranes designated for western blots were treated with goat blocking serum (Vector Laboratories) for 1 h, then incubated with rabbit antibodies against GAPDH (1:2000, Thermo Fisher Scientific), ST3Gal6 (1µl/mL) and ST6GalNAc3 (2µl/mL) (Sigma-Aldrich) for 2 h, and finally treated with WestVision Peroxidase Polymer Anti-Rabbit IgG (Vector Laboratories) for 30 min at room T°C. The color reaction on all membranes was developed after exposure to DAB Peroxidase Substrate solution (Vector Laboratories). The approximate molecular weight of the detected protein bands was estimated versus Perfect™ Tricolor Protein Ladder (EurX), ranging from 11 to 245 kDa.