NFATc1 Knockdown in Osteoclastogenesis

Cells were transfected with NFATc1 siRNA, and Non-Correlated (NC) siRNA (Qiagen, Hilden, Germany) as previously reported [17 (link)]. In brief, cells (2.5 × 10⁵) were seeded onto 6-well plates in DMEM without antibiotics; 24 h later, the transfection of siRNAs was carried out with Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA). All transfections were carried out with 20 µM duplex siRNA in medium without FBS and antibiotics. After 24 h, cells were split into 6-well plates and incubated with RANKL (50 ng/mL) for 24 and 72 h to perform further analysis. After this time, mRNA analysis was carried out by qPCR, protein analysis was performed by Western blot, and cell staining was performed by immunofluorescence. Experiments were repeated three times.