Fig. 4

EEST negatively controls F-actin structure, bone-resorbing activity, and expression of osteoclast-specific genes. a BMMs were cultured for 4 days in the presence of M-CSF (30 ng/mL) and RANKL (50 ng/mL) with or without EEST (50 μg/mL). Cells were fixed with 3.7 % formalin, permeabilized with 0.1 % Triton X-100, and stained with phalloidin and DAPI. Mature osteoclasts were seeded on hydroxyapatite-coated plates for 24 h with EEST (50 μg/mL) treatment. Adherent cells were removed and photographed under a light microscope. b The relative ratio of pit areas was quantified using Image J. ***P < 0.001 vs. control. c BMMs were stimulated with RANKL (50 ng/mL) and M-CSF (30 ng/mL) in the presence or absence of EEST (50 μg/mL) for the indicated times. Total RNA was isolated from cells using QIAzol reagent, and mRNA expression of β3-integrin, DC-STAMP, Atp6v0d2, and Cathepsin K was determined using quantitative real-time RT-PCR. ***P < 0.001 vs. control in the indicated time