Fig. 6.

Cell-derived ECM inhibited osteoclastogenesis of BMMs via attenuation of RANKL-induced ROS production. BMMs were cultured on the standard TCPS or ECM-coated substrate and treated with 20 ng/mL M-CSF and 50 ng/mL RANKL. (A) After a 24-h culture, the levels of intracellular ROS in the TCPS-cultured and ECM-cultured BMMs were measured. (B) Representative immunofluorescence staining images of the NF-κB signaling pathway. RANKL treatment induced nuclear localization of p65 in the TCPS-cultured BMMs, but failed to induce translocation in the ECM-cultured cells, indicating that ECM inhibited the activation of the NF-κB signaling pathway. Scale bar = 20 μm. (C) Western blot assay showed that cell-derived ECM inhibited NF-κB activation by suppressing phosphorylation of IκB. (D) The protein levels of p65 were evaluated in the TCPS-cultured and ECM-cultured BMMs. The GAPDH lane served as a loading control. Values are the mean ± S.E.M. of three independent experiments (n = 3). Statistically significant differences are indicated by * p < 0.05 or ** p < 0.01 between the indicated groups.