Fig 1: YKL-05–099 blocks M-CSF action.(A) CSF1R in vitro kinase assays were performed in the presence of increasing doses of YKL-05–099. This compound blocks CSF1R activity with an IC50 of 1.17 nM. (B) Murine bone-marrow-derived macrophages were grown in the presence of M-CSF, RANKL, and the indicated doses of YKL-05–099. After 3 days of differentiation, TRAP staining (purple) was performed. YKL-05–099 blocks osteoclast differentiation and causes cytotoxicity in these cultures (scale bar = 50 µm). (C) After three days of differentiation in the presence of M-CSF and RANKL, conditioned medium was collected and secreted TRAP assays were performed. YKL-05–099 treatment causes a dose-dependent reduction in TRAP secretion. Values indicate mean ± SD of n = 3 wells per condition. (D) After three days of differentiation in the presence of M-CSF and RANKL, TRAP staining was performed. The number of TRAP positive multinucleated cells (MNCs) per well of a 96-well plate (n = 3 wells/condition) is shown. (E) Murine bone marrow macrophages were grown in the presence of M-CSF for 5 days. Cells were then deprived of M-CSF for 6 hr, then pre-treated plus/minus YKL-05–099 (15 µM) for 60 min. Cells were then re-challenged with M-CSF (50 ng/ml) for the indicated times followed by immunoblotting. YKL-05–099 pre-treatment blocks M-CSF-induced M-CSFR autophosphorylation and ERK1/2 phosphorylation. (F, G) Cells as in (E) were challenged with M-CSF (50 ng/ml) for the indicated times, followed by RT-qPCR for the M-CSF target genes Ets2 and Egr1. YKL-05–099 pre-treatment blocks M-CSF-induced Ets2 and Egr1 up-regulation. (H, I) Bone marrow macrophages from ubiquitin-CreERt2; SIK2/3 (DKO) or ubiquitin-CreERt2; SIK1/2/3 (TKO) floxed mice were treated plus/minus 4-OHT as in Figures 5 and 6, then plated in M-CSF/RANKL plus the indicated doses of PLX-5622 (H) or YKL-05–099 (I) for 12 days followed by secreted TRAP assays (n = 6 wells from two independent experiments were assayed). In these plots, ‘control’ cells include BMMs from both DKO and TKO mice treated with vehicle prior to M-CSF/RANKL and inhibitor dose response. Irrespective of the cellular genotype, CSF1R inhibitor (PLX-5622 or YKL-05–099) treatment potently blocked osteoclast differentiation. All in vitro experiments were repeated three times. See Source Data File for additional information.