Increased pro-osteoclastogenic transcription factors in ASXL1-deficient osteoclasts. (A) ASXL1^flox/flox and ASXL1^cKO BMMs were exposed to M-CSF and RANKL (50 ng/mL) for 2 days. H3K27me3 binding to NFATc1 response element in the NFATc1 promoter was determined by ChIP assay. Immunoglobulin G (IgG) served as a control. (B) ASXL1^flox/flox and ASXL1^cKO BMMs were cultured in M-CSF and RANKL (50 ng/mL) for 1 day. PRC proteins were determined by immunoblot. Actin served as loading control. (C) ASXL1^flox/flox and ASXL1^cKO BMMs were cultured in the presence of M-CSF and RANKL (50 ng/mL). RNA was harvested on days 1 and 2 of RANKL stimulation, and Blimp1 mRNA abundance was determined by qPCR. (D) ASXL1^flox/flox and ASXL1^cKO BMMs were exposed to M-CSF and RANKL (50 ng/mL) for 2 days. H3K27me3 binding to Blimp1 promoter was determined by ChIP assay. IgG served as control. (E) ASXL1^flox/flox and ASXL1^cKO BMMs were exposed to M-CSF and RANKL (50 ng/mL) for 2 days. NFATc1 binding to Blimp1 promoter was determined by ChIP assay. IgG served as control. n = 3 independent experiments from 10- to 12-week-old male mice. Two-way ANOVA was used for statistical analysis. Error bars represent + standard error of the mean; **P < .01, ***P < .001.