Stable glutamine

Equid herpesvirus type 1 strain RacL11 EHV-1-RFP [9 (link)] with a red fluorescent protein (RFP) fused to the small capsid protein VP26 [3 (link)] was used in this study. The virus further expresses the enhanced green fluorescent protein (eGFP) for efficient identification of infected cells. The virus was grown on primary equine dermal (ED) cells (CCLV-RIE 1222, Federal Research Institute for Animal Health, Germany) as described before [3 (link)]. The cells were propagated in Isocove’s Liquid Medium with stable glutamine (Pan-Biotech GmbH) supplemented with 20% fetal calf serum (Pan-Biotech GmbH), 0.5% penicillin (Roth), 0.5% streptomycin (Alfa Aesar), 1% sodium pyruvate 100 mM (Pan-Biotech GmbH), and 1% nonessential amino acids (Merck KGaA). For microscopy experiments, virus was purified by ultracentrifugation over a 30% sucrose solution followed by sucrose step gradient ultracentrifugation exactly as described before [10 (link)].

In vitro and in vivo experiments were performed using the PSMA-positive C4-2 cell line, a subline of the LNCaP (lymph node carcinoma of the prostate) cell line (CRL-3314; American Type Culture Collection). C4-2 cells were cultivated in RPMI-1640 (PAN Biotech) supplemented with 10% fetal calf serum and stable glutamine (PAN Biotech). Cells were grown at 37 °C and incubated in humidified air equilibrated with 5% CO₂.

Immortalized human embryonic kidney (HEK 293) cells were commercially obtained from ATCC, catalogue Nr. CRL-1573. Cells were cultured in DMEM with GlutaMAX^TM (Gibco, Paisley, UK) or stable glutamine (PAN Biotech, Aidenbach, Germany) containing high glucose (25 mM) and 1 mM sodium pyruvate. The medium was supplemented with 50 μg/mL uridine (Sigma-Aldrich/Merck, Darmstadt, Germany), 10% heat-inactivated tetracycline-free FBS (PAN Biotech, Aidenbach, Germany), and 100 U/mL penicillin and streptomycin (Gibco, New York, NY, USA). The POLGexo^−/− cell line (p.D274A mutant) was obtained by CRISPR/Cas9 genome editing as described in Ref. [12 (link)]. The genotype was confirmed by Sanger sequencing (Figure S1).
Transient oxidative stress was induced on cells seeded at 90% confluence by applying H₂O₂ (Honeywell, Seelze, Germany) at concentrations of 0.5 or 1 mM. Viability was determined by adding 0.1% erythrosine B (Sigma-Aldrich, St. Louis, MO, USA) to cells suspended in 1× PBS (Gibco, Paisley, UK) and using a Neubauer hemocytometer (Paul Marienfeld, Lauda-Königshofen, Germany).

Human peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of the RA patients (three donors). Isolation was performed using Histopaque®-1077 (Sigma-Aldrich, Munich, Germany) as density gradient and SepMate™-50 (Stemcell™ Technologies, Vancouver, BC, Canada) tubes according to the manufacturer's protocol. The tubes were centrifuged with 1200 × g for 20 min with full acceleration and break on. After isolation, cells, now referred to as mononuclear osteoclast progenitor cells (OPCs), were cultured in cell-repellent 6-well cell culture plates in Roswell Park Memorial Institute (RPMI; PAN-Biotech, Aidenbach, Germany) 1640 supplemented with 1% amphotericin B, 1% penicillin–streptomycin, 20% FCS and 2% stable glutamine (PAN-Biotech, Aidenbach, Germany). After five days, cell supernatant with non-adherent cells was transferred to 50 ml tubes, each well of the plate was rinsed with PBS, and the tubes were centrifuged at 120 × g for 8 min.
For co-culture experiments, cells were seeded in 12-well cell culture plates with 300.000 cells per well and incubated for 14 days. For differentiation of OPCs in polynuclear osteoclast-like cells (OLCs), the medium was supplemented with 5 µg/mL human sRANK-Ligand and 2,5 µg/mL human macrophage colony-stimulating factor (M-CSF; both: Peprotech Inc., Rocky Hill, NJ, USA). After seven days of incubation, the whole medium was replaced.

Primary bone marrow cells were harvested from the tibiae and femurs of CT and SBA mice and grown for 7-10 days in αMEM media (PAN BIOTECH; P04-21050) with 15% fetal bovine serum (PAN BIOTECH; P30-3306), 1% penicillin/streptomycin (PAN BIOTECH; P04-85100) and 1% stable glutamine (PAN BIOTECH; P06-07100). After reaching full confluency, the plated BMSC population was differentiated into both osteoblasts and adipocytes according to the co-differentiation protocol described in a previously published article (30 (link)). The media containing co-differentiation inducers was replaced every two days during the 14 days of the co-differentiation experiment. This culture duration was chosen because of its brevity, which allowed for minimizing the time for cells to change after leaving the in vivo state.

Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) and fetal bovine serum (FBS) were obtained from Corning (Tewksbury, MA, USA) and Biosera (Nuaille, France), respectively. Stable glutamine and Minimum Essential Medium non-essential amino acids solutions were purchased from Pan Biotech (Aidenbach, Germany). Insulin, Ex-4, resazurin based cell viability kit (TOX-8), Triton X-100, Phosphatase inhibitor cocktail 2 were obtained from Sigma (St. Louis, MO, USA). STZ was purchased from Cayman Chemical Company (Ann Arbor, MI, USA) and was dissolved in citrate buffer (0.1 M, pH 4.5) immediately before adding to the medium. DuoSet IC, Phospho-GSK-3α/β (S21/S9) ELISA kit was provided by R&D Systems GmbH (Wiesbaden, Germany).

Monocyte isolation via positive selection was performed according to the manufacturer’s protocol StraightFrom^® Whole Blood CD14 MicroBeads (human, Miltenyi Biotec, Bergisch Gladbach, Germany). The negative selection was performed using the EasySep^TM Direct Human Monocyte Isolation Kit (STEMCELL, Vancouver, BC, Canada) as previously described [22 (link)]. After the isolation, monocytes were diluted in RPMI 1640 medium (specification: very low endotoxin), supplemented with 2 mM stable glutamine, 10% fetal bovine serum, 100 U/mL penicillin, and 0.1 mg/mL streptomycin (all: PAN-Biotech, Aidenbach, Germany). The cells were seeded in cell-repellent multiwell plates with a concentration of 1 × 10⁶ cells/ml and incubated at 37 °C and 5% CO₂ according to the applied experimental design.