Fig. 3

S. hexaphylla downregulates RANKL-induced expression of NFATc1 and c-Fos stability. BMMs were pretreated with S. hexaphylla for 1 h and then stimulated with RANKL (100 ng/mL) for the indicated times. Total RNA or protein of c-Fos and NFATc1 were obtained at the indicated time points, respectively. a mRNA expression of c-Fos and NFATc1 was analyzed by real-time RT-PCR. b Western blot analysis was performed with the indicated antibodies. β-actin was used as an internal control. c BMMs were infected with pMX-c-Fos-IRES-EGFP (c-Fos). Infected BMMs were pretreated with or without S. hexaphylla in the presence of M-CSF (30 ng/mL) for 24 h and then stimulated with RANKL (100 ng/mL). After 20 h, 2 μg/mL CHX, 5 μM MG132, and 20 μM ALLN were added to the cultures for 4 h before harvest. Western blot analysis was then performed on the cells. d BMMs were infected with pMX-IRES-EGFP (control vector), pMX-c-Fos-IRES-EGFP (c-Fos), or (e) pMX-CA-NFATc1-IRES-EGFP (CA-NFATc1). After infection, the cells were cultured in the presence of M-CSF and RANKL with or without S. hexaphylla. f TRAP-positive MNCs were counted. ^***P < 0.001; versus vehicle (DMSO). ns: not significant; ^***P < 0.001; ^*P < 0.05 versus vehicle (DMSO)