Figure 4. Cx3cr1^neg and Cx3cr1⁺ i-OCLs differ in their resorbing activity.

(A) Heatmap visualization of the z-scored expression for selected genes involved in bone resorption, osteoclast fusion and differentiation that are differentially expressed between GFP⁺ (Cx3cr1⁺) and GFP^neg (Cx3cr1^neg) i-OCLs both differentiated from WT Cx3cr1^GFP/+ mice (adjusted p-value<0.05, FC ≥2). (B) RT-qPCR analysis on GFP⁺ (Cx3cr1⁺) and GFP^neg (Cx3cr1^neg) i-OCLs. Results are represented as the mean with 95% confidence interval of 3 independent biological replicates in triplicates. (C) Flow cytometry analysis of TRAcP expression using ELF95 substrate in the 2 i-OCLs subsets. Percentage of positive cells (dark curve) compared to the negative control (light curve) is indicated. (D) Quantification of the mean fluorescence intensity (MFI) of TRAcP in GFP⁺ (Cx3cr1⁺) and GFP^neg (Cx3cr1^neg) i-OCLs (n = 3). (E) Representative images of matrix dissolution activity of sorted GFP⁺ (Cx3cr1⁺) and GFP^neg (Cx3cr1^neg) i-OCLs seeded at the same cell density. Red alizarin staining of the mineralized matrix revealed the resorbed areas as unstained. Scale bar = 100 µm. (F) Quantification of resorbed areas from three independent experiments in triplicates are indicated as percentage. ****p<0.0001; n.s., no significant difference.

Figure 4—figure supplement 1. Cx3cr1⁺ i-OCLs differ from Cx3cr1⁺ BM-derived macrophages.

(A) Schematic representation of the differentiation of macrophages from the BM of Cx3cr1^GFP/+ mice. (B) FACS analysis of macrophage markers on BM-derived macrophages and (C) on GFP⁺ (Cx3cr1⁺) and GFP^neg (Cx3cr1^neg) i-OCLs (n = 3). (D) Quantification of CD68⁺CD169⁺ cells in Cx3cr1⁺ BM-derived macrophages and in Cx3cr1^neg and Cx3cr1⁺ i-OCL subsets. (E) Representative images of TRAcP staining on i-OCLs and BM-derived macrophages showing that contrasting to i-OCLs that all express TRAcP, BM-derived macrophages are negative for TRAcP expression. Scale bar = 50 µm.