A2916801

Fibroblasts derived from Gaucher patients (GM00372 and GM00877) were purchased from Coriell Institute (Camden, NJ). Cells were maintained at 37 °C with 5% CO₂ in Minimal Essential Medium (MEM, Gibco 41500-34) supplemented with 15% fetal bovine serum (FBS, Gibco 1887826), 1% l-glutamine (Gibco A29168-01), 1% sodium pyruvate (Gibco 11360-070) and 1% non-essential amino acids (Gibco 11140050).

Mouse PSCs were cultured on Mitomycin C (M4287, Sigma-Aldrich)-inactivated mouse embryonic fibroblast (MEF) feeders in ESC medium consisting of Dulbecco’s modified Eagle’s medium with high glucose, pyruvate (DMEM, 11995065, Gibco), 15% fetal bovine serum (FBS, TMS-013-BKR, Millipore), 1,000 U/mL mLIF (ESG1107, Millipore), 1% Penicillin-Streptomycin (15140-122, Gibco), 2 mM L-glutamine (A29168-01, Gibco), MEM NEAA (11140-050, Gibco), 1 mM sodium pyruvate (11360-070, Gibco) and 0.1 mM 2-mercaptoethanol (ES-007-E, Millipore). The cells were maintained in a 37 °C humid incubator with 5% CO₂, and the culture medium was changed every two days. Retinoic acid (RA, R2625, Sigma-Aldrich), 2-deoxyglucose (2DG, D6134, Sigma-Aldrich), 6-aminonicotinamide (6AN, A68203, Sigma-Aldrich) and guanosine (G6264, Sigma-Aldrich) were used in the experiments.

Murine C2C12 myoblasts were cultured in DMEM (Dulbecco’s Modified Eagle’s Medium; 11960044; Gibco/Thermo Fisher Scientific; Waltham, MA, USA) supplemented with 10% FBS (Fetal Bovine Serum; A5256801; Gibco/Thermo Fisher Scientific; Waltham, MA, USA), 1% penicillin-streptomycin (P/S; 10,000 units/mL (P) and 10,000 µg/mL (S); 15140122; Gibco/Thermo Fisher Scientific; Waltham, MA, USA) and 1% L-glutamine (L-Gln; 200 mM; A2916801; Gibco/Thermo Fisher Scientific; Waltham, MA, USA) at 37 °C in a humidified atmosphere with 5% CO₂. The C2C12 cell line was kindly provided to us by the Poserns Lab (Martin-Luther-University Halle-Wittenberg, Institute for Physiological Chemistry; Halle (Saale), Germany). Growth medium was changed every 48 h.
To assess the effect of MGO and GO on C2C12 myoblasts, the cells were seeded at a density of 9000 cells/cm²; in a 10 cm (Ø) dish and incubated at 37 °C in a humidified atmosphere with 5% CO₂. After one day, the cells were cultured in starvation medium (DMEM + 1% FBS + 1% P/S + 1% L-Gln) with MGO or GO (0.2 mM, 0.5 mM, or 2 mM). Following another day, the cells were divided into two pellets, one for protein isolation and one for RNA isolation.

The human pancreatic cancer cell line MIA PaCa-2 (ATCC#: CRL-1420™) was used. For both in vitro and in vivo experiments, cell cultures of passages 3 to 10 and a confluence of 70–90% were used. For in vitro experiments, cultured cells were supplied with high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM, 11965092; Gibco, ThermoFisher Scientific, Waltham, MA, USA) enhanced with 10% fetal bovine serum (FBS, A5256701; Gibco, ThermoFisher Scientific, Waltham, MA, USA) and 4 mM GlutaMax (35050079; Gibco, ThermoFisher Scientific, Waltham, MA, USA). For in vivo experiments, cells were cultured in Ham’s F12 medium (21127030; Gibco, ThermoFisher Scientific, Waltham, MA, USA) enhanced with 2 mM L-glutamine (A2916801; Gibco, ThermoFisher Scientific, Waltham, MA, USA) and 10% FBS (Gibco, ThermoFisher Scientific, Waltham, MA, USA). For cell detachment from flasks, TrpyLE (12605010; Gibco, ThermoFisher Scientific, Waltham, MA, USA) was used. For cell fixation, paraformaldehyde (PFA, 047392.9M; Sigma-Aldrich, St. Louis, MO, USA) was used. For cell washing, phosphate-buffered saline (PBS, J61196.AP, ThermoFisher Scientific, Waltham, MA, USA) was used. All cell incubations were at 37 °C with 5% CO₂.

The FGSC line was cultured as described previously [23 (link), 45 (link), 46 (link)]. In brief, FGSCs were cultured on mitotically inactivated STO (SIM mouse embryo-derived thioguanine and ouabain-resistant) feeder cells at 37 °C with 5% CO2. The culture medium was minimum essential medium alpha (12000022, Gibco), supplemented with 10% fetal bovine serum (FBS03ES-5001, Front), 10 ng/mL human basic fibroblast growth factor (10018b, PeproTech), 10 ng/mL mouse glial cell line-derived neurotrophic factor (45044, PeproTech), 10 ng/mL mouse epidermal growth factor (31,509, PeproTech), 10 ng/mL mouse leukemia inhibitory factor (sc4378, Santa Cruz Biotechnology), 1 mM non-essential amino acids (11140050, Gibco), 2 mM L-glutamine (A2916801, Gibco), 50 U/mL penicillin and 50 μg/mL streptomycin (15070063, Gibco), 1 mM pyruvate (11360070, Gibco), and 100 μM β-mercaptoethanol (M3148, Sigma-Aldrich). FGSCs were subcultured every 3 or 4 days by digestion with TrypLE™ Express (12604021, Gibco) at a 1:4 or 5 split ratio. The culture medium was changed every 2 days. FGSCs were identified by RT-PCR and immunofluorescence (Additional file 1: Fig. S1).

C2C12, Sol8, and HEK-293 cells were cultured in DMEM (Dulbecco’s Modified Eagle’s Medium; 11,960,044; Gibco/Thermo Fisher Scientific; Waltham, MA, USA) supplemented with 10% FBS (Fetal Bovine Serum; A5256801; Gibco/Thermo Fisher Scientific; Waltham, MA, USA), 1% penicillin–streptomycin (P/S; 10,000 units/mL (P) and 10,000 μg/mL (S); 15,140,122; Gibco/Thermo Fisher Scientific; Waltham, MA, USA), and 1% L-glutamine (L-Gln; 200 mM; A2916801; Gibco/Thermo Fisher Scientific; Waltham, MA, USA) at 37 °C in a humidified atmosphere with 5% CO₂. For C2C12 differentiation, normal growth medium (GM) was replaced by differentiation medium (DM). This medium consists of DMEM supplemented with 2% horse serum (S-30-L; c.c.pro GmbH; Oberdorla, Germany), 1% P/S, and 1%L-Gln. HEK-293 (ACC 305) and C2C12 cells (ACC 565) were purchased from DSMZ (Braunschweig, Germany). Sol8 wild type and Sol8 Gne-knockout cells were kindly provided by Stella Mitrani-Rosenbaum (Hadassah–The Hebrew University Medical Center, Israel) (Ilouz et al. 2022 (link)). Peracetylated N-acetylmannosamine (Ac₄-ManNAc) was purchased from Synvenio (SV3881; Nijmegen, The Netherlands), N-acetylmannosamine (ManNAc) was from New Zealand Pharmaceuticals Limited (Palmerston North, New Zealand), and N-acetylneuraminic acid (Neu5Ac) was from Molekula Group GmbH (39596039-10 g; Munich, Germany).