10 cm cell culture dishes

HEK293T cells were seeded in 24 well plates (Greiner Bio-One) at a concentration of 1 × 10⁵ cells per well on a coverslip for immunofluorescence experiments or at 2 × 10⁵ cells per well for secreted embryonic alkaline phosphatase assays. For immunoprecipitation, HEK293T cells were seeded in 10 cm cell culture dishes (Greiner Bio-One) at 4 × 10⁶ cells per dish. Cells were seeded 1 day before transfection. On the day of transfection, FuGENE 6 Transfection Reagent (Roche) was added into Opti-MEM® I (1x) in GlutaMAX(TM)-I (Gibco, Life Technologies) and incubated for 5 min at room temperature before mixed with relevant plasmids according to manufacturer's instructions. Transfection mixtures were incubated for 20 min before added to seeded cells. Transfected cells were placed in 37°C incubator with 5% CO₂ for 18 h incubation before harvesting for other experiments.

hOCT3 was C-terminally YFP-tagged and single point mutations were introduced as previously described [38 (link)]. Constructs were verified by sequencing (LGC Genomics, Berlin, Germany). In brief, using jetPRIME© (Polyplus Transfection; VWR International GmbH, Vienna, Austria) reagent (for a 10 cm dish with 1–2 × 10⁶ cells in 10 mL serum containing medium at 60–80% confluency: 500 µL jetPRIME^© buffer, 10 µg DNA and 20 µL jetPRIME^© reagent), HEK293 cells were transfected with the respective plasmid containing the wildtype or variant, according to the manufacturer’s instructions (VWR International GmbH, Vienna, Austria) [39 (link)]. After selection pressure was maintained for 14 days by adding 100 µL geneticin (G418, 50 mg·mL⁻¹), 500,000 cells were FACS-sorted (fluorescence-activated cell sorting) according to expression levels to establish polyclonal cell lines. In cell culture, cells were maintained at high glucose (4.5 g·L⁻¹), l-glutamine-containing (584 mg·L⁻¹) Dulbecco’s Modified Eagle Medium (Sigma-Aldrich, St. Louis, MO, USA) with 10% heat-inactivated foetal bovine serum (FBS, Sigma-Aldrich), penicillin (1 U·mL⁻¹, Sigma-Aldrich) and streptomycin (1 µg·mL⁻¹, Sigma-Aldrich). To maintain selection pressure the medium was supplemented with 50 µg·mL⁻¹ geneticin (G418). Cells were maintained in 10 cm cell culture dishes (Greiner) at 37 °C and 5% CO₂ in a humidified incubator.

Activity of NF-κB was determined in DFCs seeded in 10-cm cell culture dishes (#664160 from Greiner Bio-One) after 7 days of osteogenic induction. Nuclear proteins were enriched and isolated from DFCs as described in the Western blot analysis section. Activity of NF-κB subunits p50 and p65 was then determined with the TransAM® NF-κB Family Kit (Active Motif, Carlsbad, USA) according to the manufacturer’s instructions.