Nucleotides

Methanol, ultrapure water, formic acid, sodium chloride, potassium chloride, and ammonium acetate were purchased from Fisher Chemical. Nafion perfluorinated resin solution (20 wt% in lower aliphatic alcohols and water), trypsin, insulin, cytochrome c, N-acetyl neuraminic acid, nucleotides, and nucleoside standards were all purchased from Sigma-Aldrich. Polymethyl methacrylate (PMMA, molecular weight within 30–90 kDa) and multi-walled carbon nanotube (MWCNT, ID 2–5 nm, OD < 8 nm, length 10–30 μm) were purchased from Adamas-beta Reagent Ltd. and J&K Scientific, respectively. Ultracentrifuge spin columns (cutoff molecular weight: 3.0 kDa) were purchased from Millipore. The LTQ Orbitrap Velos mass spectrometer (Thermo Scientific, San Jose, CA) was employed for recording the mass spectra data.

Aprotinin, nucleotides, apyrase grade VII, phenylmethanesulfonyl fluoride (PMSF), ticlopidine, and malachite green were purchased from Sigma-Aldrich (Oakville, ON, Canada). Tris was obtained from VWR (Montreal, QC, Canada). DMEM was obtained from Invitrogen (Burlington, ON, Canada). Fetal bovine serum (FBS) and antibiotics-antimycotics solution were from Wisent (St-Bruno, QC, Canada). Formalin and acetone were obtained from Fisher Scientific (Ottawa, ON, Canada). OCT freezing medium was purchased from Tissue-Tek, Sakura Finetk (Torrance, CA).

Folate, amino acids, nucleosides, nucleotides and other chemical compounds were purchased from Sigma-Aldrich (St. Louis, MO, USA). Minimal essential medium/alpha modified (αMEM) without ribosides, ribotides, deoxyriboside, deoxyribotides, glycine, serine and Folate was specially ordered and formulated by Invitogen and JRH (Lenexa, KS, USA). Fetal bovine serum (FBS) was from Biological Industries (Kibbutz, Israel). Penicillin, streptomycin, fungizone, trypsin and trypan blue were from GIBCO Laboratories (Grand Island, NY, USA).
Antibodies specific for heavy subunit of γ-glutamylcysteine synthetase (γ-GCS_h), COX-2, Bcl-2, GRP78, ATF4, ATF6α, SP-1 (Santa Cruz Biotechnology, california, USA), Survivin, pro-caspase 3 (Cell Signaling Technology, Danvers, MA, USA), PARP-1, β-catenin (Epitomics, california, USA), and β-actin (Sigma-Aldrich, St. Louis, MO, USA) were obtained.

Nucleotides, doxorubicin, anti-GFP antibody, benzamidine, poly-d-lysine, saponin, and 2-mercaptoethanol were purchased from Sigma Aldrich (St. Louis, MO, USA). Estradiol glucuronide (E217βG) and sodium orthovanadate were obtained from Santa Cruz (Dallas, TX, USA). MK571 was received from Cayman Chemicals (Ann Arbor, MI, USA). Restriction enzymes were purchased from New England Biolabs (New England, MA, USA) and phosphate-buffered saline (PBS) was purchased from Thermo Scientific (Sunnyvale, CA, USA).

Verapamil hydrochloride was purchased from Shanghai Hefeng Pharmaceutical Co., Ltd. (Shanghai, China). Barium chloride was obtained from Chongqing Maoye Chemical Reagent Co., Ltd. Optimal LC grade acetonitrile was obtained from Merck (Merck, Darmstadt, Germany). Isopropanol, formic acid and nucleotides for metabolite analyses were purchased from Sigma-Aldrich (Spruce St., St Louis, MO, United States).

Myoblasts were cultured on glass coverslips in 35 mm diameter dishes for 48 h under the conditions described above. Cells (70–80% confluent) were loaded with 2 μM Fura 2 AM (MolecularProbes, Oregon) in the serum-free culture medium for 20 min at 37 °C in a humidified atmosphere of 95% O₂ and 5% CO₂. The cells were then washed twice with the solution composed of 5 mM KCl, 1 mM MgCl₂, 0.5 mM Na₂HPO₄, 25 mM HEPES, 130 mM NaCl, 1 mM pyruvate, 5 mM d-glucose, and 0.1 mM CaCl₂, pH 7.4 and the coverslips were mounted in a cuvette containing 3 ml of the nominally Ca²⁺-free assay solution (as above but 0.1 mM CaCl₂ was replaced by 0.05 mM EGTA) and placed (at RT) in a spectrofluorimeter (Shimadzu, RF5001PC). The cells were treated with nucleotides (Sigma) applied at a concentration indicated in the relevant figures and 10 μM AR-C 118925XX (216657-60-2, TOCRIS Bioscience) applied 10 min before the addition of agonists. Fluorescence was recorded at 510 nm, with excitation at 340 and 380 nm. At the end of each experiment, the Fura 2 fluorescence was calibrated by the addition of 33 μM ionomycin to determine maximal fluorescence, followed by the addition of EGTA to complete the removal of Ca²⁺. Cytosolic Ca²⁺ concentration [Ca²⁺]c was calculated according to Grynkiewicz et al.^{38 (link)}.