Sodiumpyruvat

SEM (55 (link)), Jurkat, CEM, MOLT-16 and Nalm-6 cells (DSMZ) were cultured in RPMI 1640 GlutaMax-I medium (Thermo Fisher Scientific) supplemented with 10% fetal calf serum (FCS; Thermo Fisher Scientific), 100 U/mL penicillin and 100 µg/mL streptomycin (Thermo Fisher Scientific) (R10+). BHK-CD16a (FcγRIIIa V158) cells were cultured in R10+ medium supplemented with 10 μmol/l methotrexate (Sigma-Aldrich) and 500 μg/ml geneticin (Thermo Fisher Scientific) as described before (53 (link), 54 (link), 56 (link)). CHO-CD32a (FcγRIIa H131) cells were cultivated in DMEM medium (Thermo Fisher Scientific) supplemented with 10% FCS, 100 U/ml penicillin and 100 µg/ml streptomycin (57 (link)). Medium was supplemented with 1% NEAA (Thermo Fisher Scientific), 1% sodiumpyruvat (Thermo Fisher Scientific) and 500 µg/ml geneticin. Chinese hamster ovary cells (CHO-S, Thermo Fisher Scientific) were cultured in serum-free CD-CHO medium (Thermo Fisher Scientific) containing 1% HT supplement (Thermo Fisher Scientific) and 2 mM GlutaMax (Thermo Fisher Scientific). After transfection CHO-S cells were cultured in CD OptiCHO medium (Thermo Fisher Scientific) containing 1% HT supplement, 2 mM GlutaMax and 0.1% Pluronic F-68 (Thermo Fisher Scientific).

The human cell lines HEK293T (embryonic kidney) and Colo320 (colon cancer) were maintained in Dulbecco’s modified eagle medium (DMEM, Thermo Fisher Scientific), supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific), 100 U/ml penicillin and 100 µg/ml streptomycin (Thermo Fisher Scientific) in a humidified atmosphere containing 5% CO₂ at 37 °C. Transfections of HEK293 and Colo320 cells with Pre-miR miRNA precursors were performed in Opti-MEM I Reduced Serum Media (Thermo Fisher Scientific) using Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific). Co-transfection of HTR4 constructs and Pre-miR miRNA precursors for luciferase assays and In Cell Western (ICW) experiments were carried out with polyethylenimine (PEI, Sigma-Aldrich, St. Louis, Missouri).
Transfections and harvesting of cells are described in detail in the Supplementery Methods.
The HT29-MTX-E12 cell line (kindly provided by Dr. Marguerite Clyne, University College Dublin) was cultured in DMEM, high Glucose, GlutaMAX plus 10% FCS, 1% NEAA and 1% Sodiumpyruvat (Thermo Fisher Scientific). Transfection of miRNA precursors was performed with Lipofectamine 2000 (Thermo Fisher Scientific) and CombiMag (OZBiosciences, Marseille, France) as outlined in Supplementary Methods. Pre-miR miRNA precursors are given in Supplementary Table S5.

Primary human chondrocytes were isolated from OA cartilage samples and cultured with supplemented high glucose Dulbecco’s Modified Eagle’s Medium (DMEM) (PAA Laboratories, Pasching, Germany) at 37 °C and 5% CO₂ [41 (link)]. For nucleotide internalization P1 chondrocytes were analyzed. For all other experiments P2 chondrocytes were used. DMEM was supplemented with 10% fetal calf serum (Biochrom, Berlin, Germany), 1% sodium pyruvat (Thermo Scientific, Waltham, USA) and 1% penicillin/streptomycin/fungizone (PAA Laboratories, Pasching, Germany). Primary murine chondrocytes were isolated from neonatal C57BL/6 J wildtype mice according to Gosset et al. [42 (link)].