Dmem f12

U87 cells were maintained in DMEM/F-12 (Lonza, Verviers, Belgium) supplemented with 1% fetal bovine serum (FBS, Sigma-Aldrich, Munich, Germany) and 1% penicillin/streptomycin solution (pen/strep, Lonza). Human dermal microvascular endothelial cells (HMEC-1) were kindly provided by Dr. E.W. Ades (CDC, Atlanta, GA, USA) [49 (link)] via Prof. G. Molema and the UMCG Endothelial Cell Facility and maintained in M-199 medium (Lonza) supplemented with 10% FBS, 10% human serum (Sigma-Aldrich), pen/strep and L-glutamine (Lonza). GSCs were maintained in DMEM/F12 (Lonza) supplemented with 10% B27 (Life Technologies, Bleiswijk, the Netherlands), 20 ng/ml bFGF and EGF (Life Technologies) and pen/strep. Carrier-free recombinant human VEGFA₁₆₅ (from here on referred to as VEGFA), Ang-2 and IL-8 were obtained commercially (R&D Systems, Minneapolis, MN, USA).

For induction of muscle cell differentiation, the proliferation medium SkBM was replaced with a differentiation medium, DMEM:F12 (Lonza, Walkersville, MD, USA) with supplementation of 2% horse serum (ATCC, Baltimore, USA). The differentiation medium was changed every two days until the desired day of differentiation for parameter measurement.

Mouse C2C12 myoblast cells (ATCC: CRL-1772) were grown in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO) containing 10% fetal bovine serum (FBS; Lonza) and 1% penicillin/streptomycin (GIBCO). To induce myotube differentiation, cells over 80% confluences were cultured in differentiation media (DMEM supplemented with 2% horse serum and 1% penicillin/streptomycin). Human skeletal myoblast cells (HSMMs), human primary cells isolated from normal donors, were purchased from Lonza and cultured in SkGM-2 media (Lonza). For differentiation conditions, cells were replaced with DMEM/F12 (Lonza) with 2% horse serum. HEK293A and 293T cells (ATCC) were maintained in DMEM (GIBCO) containing 10% FBS with 1% penicillin/streptomycin. Cells were cultured at 37 °C humidified 5% CO₂ atmosphere.

Cells (1,500 cells/mL) were cultured for 8 days in serum-free DMEM-F12 (BioWhittaker, Radnor, PA, USA) supplemented with B27 (1:50; Invitrogen), 20 ng/mL EGF (Invitrogen), 20 ng/mL bFGF (Invitrogen), and anti-mycoticantibiotic (1:100; Invitrogen) in suspension in 6-well plates (Costar 3471; Corning, Corning, NY, USA).

hTERT-RPE1 cells were maintained in DMEM-F12 (LONZA), supplemented with 10% foetal bovine serum (FBS), sodium bicarbonate and penicillin/streptomycin. Cells were transfected with Lipofectamine 2000 (Life technologies) according to the manufacturer’s instructions. Briefly, cells were plated at a density of 1 X 10⁵ cells per well on a 12-well plate (Greiner Bio-One) the day prior to transfections. For immunostaining experiments cells were seeded on coverslips. One hour before transfection, growth medium was replaced by medium without antibiotics. Scrambled control and RPGR/RPGRIP1/RPGRIP1L specific siRNAs were diluted in OptiMEM (Gibco). 1.5μl of Lipofectamine-2000 was diluted in OptiMEM and incubated at room temperature (RT) for 5min. Both the diluents were mixed, incubated for 20 min, added drop wise to the cells and then incubated.

All cell culture experiments were performed in five replicates. Four replicates were used for mass spectrometric metabolome analysis and one was used to verify the knockdown efficiency by western blot. For each condition 0.5 × 10⁶ cells were seeded onto 100 mm × 20 mm cell culture dishes (Corning, Kaiserslautern, Germany), containing 10 mL DMEM/F12 (Lonza, Cologne, Germany) media supplemented with 10% foetal bovine serum (Day −1). Cells were allowed to attach overnight before treatment. Media were changed regularly starting treatment with 0.125 µg/mL tetracycline (Sigma-Aldrich, Munich, Germany) at day 0, 2, 4 and 6, respectively (Supplementary Table S1).
At day 7, cells were washed twice with 0.9% sodium chloride (Sigma-Aldrich, Munich, Germany). Metabolism was quenched by addition of 1.5 mL ice-cold extraction buffer (90% methanol in water) containing 1 µg/mL β-phenylglucose, ribitol and 5 µg/mL isoguanosine as internal standards (Sigma-Aldrich, Munich, Germany) for 1 min on ice. Cells were lysed by extensive scraping with Corning® cell scrapers (Sigma-Aldrich, Munich, Germany). Cell extracts were collected in screw-cap tubes (Sarstedt AG, Nürnbrecht, Germany) containing 300 mg glass beads (Sigma-Aldrich, Munich, Germany) and stored in liquid nitrogen immediately^{26 (link)}.

At necropsy, the joint space was exposed under a dissecting microscope. Synovial tissue was dissected out from the joint and placed in a petri dish containing PBS and 1% anti-anti (Life Technologies). Grooves/scratches were made in the bottom of a 12 well plate with a scalpel to improve tissue transfer efficiency and to promote tissue adherence. Tissue samples were transferred to the 12 well plate and MSC expansion medium, consisting of DMEM/F-12 (Biowhittaker), 10% fetal bovine serum (Life Technologies), 1% non-essential amino acid (Life Technologies), 1% anti-anti (Life Technologies), and 0.2% beta-mercaptoethanol (Life Technologies) was added to each well. Cells were then incubated at 37 °C in 2% O₂ and 5% CO₂. Medium was changed every 2–3 days until outgrowth was observed from the tissue, after which medium changes were performed daily. When cells that had outgrown from the tissue had reached confluency, the tissue piece was removed and the cells were passaged into a T25 flask. Subsequent medium changes occurred every 2–3 days, or as required.