ASXL1 deletion in myeloid lineage cells promotes osteoclastogenesis. (A) Six- to 8-week-old WT BMMs were exposed to M-CSF and RANKL (50 ng/mL) for 5 days. ASXL1 mRNA expression was determined by qPCR. One-way ANOVA was used to determine statistical differences. Data are represented as + standard deviation (SD). ***P < .001 relative to BMM control. (B) ASXL1^flox/flox and ASXL1^cKO BMMs were cultured with M-CSF + RANKL (50 ng/mL) for 5 days. ASXL1 protein was determined by immunoblot. (C) Representative image showing ASXL1^flox/flox and ASXL1^cKO BMMs cultured in M-CSF and RANKL (25 ng/mL and 100 ng/mL) for 5 days, after which cells were stained for TRAP activity. The images were captured using Nikon Eclipse E400 upright microscope. Scale bar represents 400 μm. (D) TRAP positive osteoclasts were then counted. Unpaired nonparametric Student t test was used to determine statistical differences. Error bars represent + SD; *P < .05, **P < .01 in comparison with their respective controls. (E) ASXL1^flox/flox and ASXL1^cKO BMMs were cultured with M-CSF and RANKL (50 ng/mL) for 4 days. Total cell lysate was collected with time. Osteoclast differentiation proteins were determined by immunoblot. (F) ASXL1^flox/flox and ASXL1^cKO BMMs were cultured with M-CSF and RANKL (100 ng/mL) on bovine bone slices. After 5 days, the cells were stained with Alexa-Fluor-546-phallodin to visualize the actin rings (top). The images were captured on the green channel of Nikon Eclipse E400 upright microscope. Following removal of the transduced osteoclasts, resorption pits were visualized by wheat germ agglutinin-lectin staining (bottom). Scale bar represents 100 μm. (G) ASXL1^flox/flox and ASXL1^cKO BMMs were cultured with M-CSF and RANKL (100 ng/mL) for 6 days on bovine bone slices. Conditioned medium was assayed for CTx (6 bone slices were used for both genotypes). Error bars represent + SD; **P < .01. All experiments were conducted at least 3 times.