T 75 tissue culture flasks

The murine skeletal muscle cell line, i.e., C2C12 myoblasts from American Type Culture Collection, was maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific, USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) containing 100 U/ml penicillin and 100 μg/ml streptomycin in the presence of 5% CO₂ at 37°C. The cells were seeded in T-75 tissue culture flasks (BD Falcon, USA) and used between passages 2 and 3. After reaching approximately 70% confluence, the cells were switched to differentiation medium composed of high-glucose DMEM with 2% horse serum (Thermo Fisher Scientific) in order to induce myoblast fusion into differentiated myotubes for 48 h. The myotubule formation was monitored by the use of a phase-contrast microscope (see Fig. S1 in the supplemental material).
Differentiated C2C12 myotubes were treated with 2-AA (Enamine Ltd., Ukraine) (200, 400, and 800 μM) at different time points. For the inhibitor assay, cells were pretreated with allopurinol (Abcam, USA) (50 μM), apocynin (Abcam) (10 μM), dorsomorphin (Abcam) (10 μM), gp91ds-tat (Anaspec Inc.) (50 μM), MG132 (Sigma-Aldrich) (10 μM), or NAC (Sigma-Aldrich) (5 mM) for 1 h before 2-AA treatment.
Cell viability was assessed by the MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide]; Sigma-Aldrich) assay as previously described (30 (link)).

The parental human telomerase-immortalized corneal epithelial (hTCEpi) cell line has been characterized previously [14 (link)]. We obtained this cell line from Dr. Jester’s laboratory at the University of California Irvine. hTCEpi cells were routinely maintained in serum-free keratinocyte growth media (KSFM) (with 0.15 mM Ca²⁺) supplemented with bovine pituitary extract (BPE), insulin, hydrocortisone, penicillin, streptomycin, amphotericin B and human epidermal growth factor (hEGF) (KGM™-CD from Lonza Clonetics, Walkersville, MD). Unless specified elsewhere, cells were passaged every 4 days using 0.25% trypsin-EDTA and 0.25 mg/ml soybean trypsin inhibitor (Invitrogen, Carlsbad, CA) and cultured in T75 tissue culture flasks (Falcon Labware; BD Biosciences, Bedford, MA) incubated at 37°C in 5% CO₂.

Purified CD3⁺ T cells (0.5 x 10⁶/mL) were suspended in RPMI-1640 medium, supplemented with 10% FCS (ThermoFisher Scientific; Waltham, MA), 10 μg/mL Concavalin-A and 0.22 nM (60 IU/mL) IL-2, seeded in T75 tissue culture flasks (BD Biosciences), and cultured for 4 days. At the end of this culture, activated T cells were 98% CD3⁺CD56^-CD14^-CD19^- (data not shown).

The pancreatic cancer cell lines MiaPaCa-2 and BxPC-3 were purchased from American Type Culture Collection (ATCC). MiaPaCa-2 cells were grown in DMEM (Cellgro, Manassa, VA) supplemented with 10% (v/v) fetal bovine serum (Cellgro). BxPC-3 cells were cultured in RPMI (Cellgro), 10% (v/v) fetal bovine serum and 1% (v/v) sodium pyruvate (Gibco, Grand Island, NY). Cells were maintained in 5% CO₂ at 37°C with 95% humidity and grown as monolayers in T75 tissue culture flasks (BD Biosciences, Bedford, MA).