Figure 2: CIE suppresses RANKL-induced c-Fos and NFATc1 expression. (a) Effects of CIE on levels of c-Fos and NFATc1 protein expression were evaluated using western blot analysis. β-Actin was used as the internal control. (b) BMMs were stimulated with RANKL (100 ng/mL) and M-CSF (30 ng/mL) in the presence or absence of CIE (50 μg/mL) for the specified times. Total RNA was isolated from cells using QIAzol reagent and mRNA expression levels of c-Fos and NFATc1 were evaluated using real-time PCR. versus the control at 48 h; versus the control at 12 h. (c) BMMs were infected with retroviruses expressing pMX-IRES-EGFP (pMX), pMX-NFATc1-EGFP, or pMX-cFos-EGFP. Infected BMMs were cultured with or without CIE (50 μg/mL) in the presence of M-CSF (30 ng/mL) and RANKL (100 ng/mL) for 4 days. After culturing, the cells were fixed and stained for TRAP (top). The TRAP-positive multinucleated osteoclasts were counted (bottom). .