Phalloidin conjugate solution

Osteoclastic actin rings were detected by using phalloidin staining assay. Briefly, the cultured cells were fixed with 3.7% formaldehyde in PBS for 10 min. The fixed cells were permeabilized with 0.1% Triton X‐100 for 10 min and stained with phalloidin conjugate solution (Sigma‐Aldrich) for 40 min. Then, the fixed cells were washed with PBS and the nuclei were stained with Hoechst 33342 (Sigma‐Aldrich). Fluorescence microscope was used for photomicrographs and distributions of actin rings (KS400; Carl Zeiss).

α‐Minimal essential medium (α‐MEM), penicillin, streptomycin, foetal bovine serum (FBS), qPCR SuperMix UDG kit were purchased from Invitrogen (Carlsbad, CA). Lipopolysaccharides (LPS), macrophage monocyte colony‐stimulating factor (M‐CSF), methylthiazolyldiphenyl‐tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), TRAP solution, Acid Phosphatase Liquicolor Assay kit, Hoechst 33342, ELISA kits, alamar blue reagent and phalloidin conjugate solution were purchased from Sigma‐Aldrich. Anti‐NFATc1 (H‐110), anti‐Ap1 (Sc‐57761) and anti–c‐Fos (H‐125) monoclonal antibodies were purchased from Santa Cruz Biotechnology. Anti–TNF‐α, anti‐RANKL, anti‐OPG, anti–IL‐1β, anti–IL‐6 and anti–TGF‐β were purchased from Cell Signaling Technology. NucleoSpin for RNA extraction kit was purchased from Macherey–Nagel. Toluidine blue stain was purchased from BioGenex. FastStart Essential DNA Green Master was purchased from Roche. Goat anti‐rabbit horseradish peroxidase conjugate was purchased from Bio‐Rad.