Figure 3: CIE downregulates RANKL-induced early signals and marker genes during osteoclastogenesis. (a) BMMs were pretreated with DMSO (control) or CIE (50 μg/mL) for 1 h in the presence of M-CSF (30 ng/mL) and were stimulated with RANKL (100 ng/mL) for the indicated times. Whole-cell lysates were used for western blot analysis with the specified antibodies. β-Actin served 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 indicated times. Total RNA was isolated from cells using QIAzol reagent and the mRNA expression levels of OSCAR, TRAP, integrin αv, β3, DC-STAMP, OC-STAMP, cathepsin K, and ICAM-1 were evaluated by real-time PCR. , , and versus the control at 12 h, 24 h, and 48 h.