Gene silencing of OGT and NFAT isoforms using siRNA

16HBE cells were used for NFAT and OGT siRNA-mediated knockdown (KD) experiments as previously described (5, 25). Briefly, 5 × 10⁴ cells were seeded in coated 24-well plates and transfected for 6 h in 0.5 mL OptiMEM with 5 nmol of either AllStar negative control or OGT siRNAs (Thermo Scientific, Grand Island, NY, USA) or NFATC2 (NFAT2c) or NFATC3 (NFAT3c) siRNAs (Qiagen; Hilden, Germany) using 1.5 μL/well of Qiagen HiPerFect transfection reagent. Following the transfection, medium was replaced with complete medium and the cells were subjected to an additional 48 h incubation to allow for NFAT or OGT knockdown. After a 24 h treatment with FGF23, wells were washed with 1.0 mL cold phosphate buffered saline (pH 7.4) and RNA was extracted using the GeneJET RNA purification kit (Thermo Scientific, Grand Island, NY, USA). For some experiments, HBECs were transfected with siRNA against OGT or NFAT2c/3c and 100 ul of conditioned medium was collected for ELISA.