P27kip1

For Western blot assays, conducted as previously described [7 (link)], antibodies against the following proteins were used: KDM4A (Novus, Littleton, CO, USA, NB110–40585), KDM4B (Bethyl Laboratories, Montgomery, TX, USA, A301–478A), KDM4C (Novus, NBP1–49600), KDM4D (Abcam, Hong Kong, China, ab198209), AR (Santa Cruz, sc-7305), histone H3 trimethyllysine 9 (H3K9me3) (Abcam, ab8898), histone H3 trimethyl lysine 4 (H3K4me3) (Abcam, ab8580), H3 (Abcam, ab1791), c-myc (Santa Cruz, sc-789), and p27^kip1 (Santa Cruz, sc-508). Protein bands were visualized by enhanced chemiluminescence (Pierce Biotechnology, Rockford, IL, USA), and protein expression was normalized against β-actin (Cell Signaling Technology, Danvers, MA, USA).

Cells or tissues were lysed in RIPA buffer, supplemented with protease and phosphatase inhibitors, and in some instances prepared for cytosolic and nuclear fractionation, in accordance with manufacturer's protocols (Thermo Scientific). Immunoprecipitation was performed on whole-cell lysates from 3T3-L1 adipocytes at different stages of differentiation with established protocols14 (link). Proteins were resolved by SDS–PAGE for detection, and PVDF membranes were probed with antibodies against 14-3-3ζ (#7413), C/EBP-α (D56F10, #8178), C/EBP-β (#3082), C/EBP-δ (#2318), Foxo1 (L27, #9454), Lipin1 (#5195), Pparγ (81B8, #2443), cleaved caspase-3 (5A1E, #9664), Pgc1-α (#4259), tubulin (#2146) and Lamin A/C (4C11, #4777) (all antibodies diluted 1:1,000, Cell Signaling Technology); p27^Kip1 and Gli3 (all antibodies diluted 1:200; Santa Cruz Biotechnology, Santa Cruz, CA); TAP (CAB1001) and Gli3 (PA5-28029) (all antibodies diluted 1:1,000, Thermo Scientific) and β-actin (AC-15, #NB600-501) (Novus Biologicals, Littleton, CO). Original scans of immunoblots are shown in Supplementary Fig. 8.

For analysis of cell cycle regulators or osteoclast marker proteins, cells incubated in culture medium with or without M-CSF (30 ng/mL) and then exposed (or not) to M-CSF and RANKL for the indicated times were lysed and subjected to immunoblot analysis with antibodies to cyclin A, to cyclin D1, to cyclin E, to CDK2, to CDK4, to p21^Waf1/Cip1, to p27^Kip1, to Ser¹⁰-phosphorylated histone H3, to α-tubulin, to NFATc1, to Atp6v0d2, to integrin β3, and to β-actin (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA); with those to histone H3 and to cathepsin K (Abcam, Cambridge, MA, USA); and with those to c-Fos (Cell Signaling Technology, Boston, MA, USA).

Hydrangenol was purchased from Coresciences Co. (Seoul, South Korea). Antibodies against CDK2, CDK4, cyclin D1, cyclin E, p21^WAF1, p27^KIP1, p53, and GAPDH were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Total ERK, p38, JNK, AKT, phospho-ERK, phospho-p38, phospho-JNK, and phospho-AKT antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The polyclonal MMP-9 antibody was purchased from Chemicon (Temecula, CA, USA). The nuclear extract kit and the electrophoretic mobility shift assay (EMSA) gel shift kit were obtained from Panomics (Fremont, CA, USA). The p38-specific inhibitor SB203585 was purchased from Calbiochem (San Diego, CA, USA).