FIGURE 6.

Flavopiridol treatment prevents osteoclastogenesis by targeting RANKL-induced NF-κB pathway. (A) BMMs were treated with 50 ng/mL RANKL in the presence or absence of 200 nM flavopiridol for 0-, 5-, 10-, 20-, 30-, and 60-min. The expression of total and phosphorylated p65, ERK, and β-actin was determined by Western blot. Images of bands were grouped together. (B–E) The intensity of each protein band was quantified and normalized to β-actin using Image J (n = 3, ^∗P < 0.05). (F) BMMs were treated with CDK9 inhibitor (200 nM), M-CSF (30 ng/mL), and RANKL (50 ng/mL) for 1 h and total and phosphorylated p65 and ERK was detected by immunofluorescence imaging. Nuclear positions were marked by DAPI. (G–J) The fluorescence intensity of p65 and ERK in different samples was quantified with Image J. The signal of RANKL only samples was set to 100%. The nucleus VS cytoplasm signal intensity ratio of p-P65 and p-ERK was calculated to indicate the nucleus translocation (results were the mean ± standard deviation, ^∗P < 0.05).