NFATc1 Knockdown in Osteoclastogenesis

Cells were transfected with NFATc1-siRNA or non-correlated (NC) siRNA (Qiagen, Germantown, Maryland, USA) as previously reported [13 (link)]. In brief, cells (2.5 × 10⁵) were seeded onto 6-well plates in medium without antibiotics; 24 h later, the transfection of siRNAs was carried out with Lipofectamine RNAiMAX (Invitrogen, Thermo Fisher Scientific Carlsbad, CA, USA). All transfections were carried out with 20 μM duplex siRNA in medium without FBS or antibiotics. Then, 6 h later, we added RANKL (50 ng/mL) to the medium. One or two days after transfection, cells were recovered to perform further analyses. Experiments were repeated three times.