TLR1/2 agonist treatment suppresses the osteoblast niche. Wild-type or Col2.3-GFP⁺ mice were treated with PAM₃CSK₄ (100 μg, every other day × 3 doses) and analyzed 24 hours after the final dose. (A) RNA expression from total bone marrow of the indicated gene relative to β-actin. (B) Representative flow plots showing gating strategy to identify Col2.3-GFP⁺ CD140a⁺ CD31^– lineage^– osteoblasts (Obs). (C) Quantification of Ob numbers by flow cytometry. (D) Representative immunofluorescence staining images of femur sections showing Col2.3-GFP (green) and 4′,6-diamidino-2-phenylindole (DAPI; blue). (E) Ob number per millimeter bone perimeter. (F) Quantification of osteoblast thickness. (G) RNA expression of the indicated gene relative to β-actin from sorted Col2.3-GFP⁺ CD140a⁺ CD31^– lineage^– Obs. (H) Representative flow plots of bone marrow mesenchymal stem cells (MSCs; GFP^– CD140a [PDGFRα]⁺ Sca1⁺ CD31^– lineage^–) and non-osteoblastic stromal cells (GFP^– CD140a⁺ Sca1^– CD31^– lineage^–). Quantification of MSCs (I) and non-osteoblastic stromal cells (J) by flow cytometry. Data represent the mean ± standard error of the mean. Statistical significance determined by using unpaired t-test. ∗P < .05, ∗∗P < .01, ∗∗∗∗P < .001. Ctrl, control; SSC-A, side scatter area.