Figure 3.

CEP attenuated RANKL-induced osteoclast formation in vitro. (A) BMMs were seeded at a density of 2 × 10⁴ cells/well and treated with the indicated concentrations of CEP in the presence of 30 ng/mL M-CSF for 48 or 96 h. The cell viability was quantified using the CCK8 assay. (B) TRAP staining was performed on BMMs treated with different doses of CEP in osteoclastogenic medium for 4 days. Scale bar = 200 μm. (C,D) The number and area of TRAP positive osteoclasts were analyzed. (E–J) The mRNA expression levels of Cathepsin K, TRAP, CTR, V-ATPase a3, V-ATPase d2, and DC-STAMP in BMMs treated with the indicated concentrations of CEP for 4 days were quantified. K BMMs were treated with 0.5 μM CEP for day 0–day 2 (Early-stage), day 2–day 4 (Late-stage), or day 0–day 4 (Early + Late stage) in osteoclastogenic medium. Scale bar = 200 μm. (L,M) The number and the area of osteoclasts were measured. All experiments were repeated independently for three times. Values are expressed as mean ± SD; ^*P < 0.05, ^**P < 0.01 vs. the control group.