Fig. 5: Impaired osteogenic specification of Cxcr4¹⁰¹³-bearing skeletal stromal/stem cells.

A Ki-67 and DAPI co-staining to analyze by flow cytometry the cell cycle status of SSCs and OPCs from bone fractions. Bar graphs show the percentage of cells (DAPI^lowKi-67^-) in the quiescent G0 phase. Data (means ± SEM) are from three independent experiments with n = 9, 6, and 6 mice in total for WT, +/1013, and 1013/1013 groups, respectively. Statistics were calculated with the nonparametric Kruskal–Wallis H test (^#p = 0.029) and the unpaired two-tailed Student’s t test (1013/1013 vs WT **p = 0.0091). B Flow-cytometric detection of BrdU staining in SSCs (left). Percentages of BrdU⁺ bone SSCs and OPCs after a 12-day labeling period (right). Data (means ± SEM) are from three independent experiments with six mice in total per group. Statistics were calculated with the nonparametric Kruskal–Wallis H test (^##p = 0.0021) and the unpaired two-tailed Student’s t test (+/1013 vs WT *p = 0.016, 1013/1013 vs WT **p = 0.0011). C Characterization of some biological processes displaying differential gene expression signatures in sorted SSCs as defined by GSEA and obtained by RNA-seq on 2 (1013/1013) or 3 (WT and +/1013) biological replicates per group with one replicate representing the pool of 3 mice. For significance testing, DESeq2 uses a Wald test (p values). The Wald test P values from the subset of genes that pass an independent filtering step, are adjusted for multiple testing using the procedure of Benjamini and Hochberg (padj values). D RNA-seq-based heatmap representing the relative expression levels of osteogenic genes. E Normalized counts of selected osteogenic genes using the DESeq2 method. Data are represented as floating bars (min to max and line equal median) of the 2 or 3 biological replicates per group. For significance testing, DESeq2 uses a Wald test (p values). F The heatmap shows the relative expression levels (RQ) normalized for β-actin expression levels in each sample of selected genes involved in SSC differentiation towards the osteogenic lineage (6 pools of 100 cells per condition) by quantitative PCR. G RQ of the most regulated genes involved in differentiation and cell cycle of SSCs. Data (means ± SEM) are from two independent experiments with 6 mice in total per group. Statistics were calculated with the nonparametric Kruskal–Wallis H test (^#p = 0.028 for Runx2; ^#p = 0.011 for Ccnd3) and the unpaired two-tailed Student’s t test (1013/1013 vs WT **p = 0.0063 for Runx2; 1013/1013 vs WT *p = 0.048 for Col1α; +/1013 vs WT *p = 0.022 and 1013/1013 vs WT *p = 0.02 for Ccnd3). H RNA-seq-based heatmap representing the relative expression levels of osteoclastogenic genes expressed by sorted SSCs. I Normalized counts of selected osteoclastogenic genes using the DESeq2 method. Data are represented as floating bars (min to max and line equal median) of the 2 or 3 biological replicates per group. For significance testing, DESeq2 uses a Wald test (p values). J Relative expression of osteoclastogenic genes in SSCs by quantitative PCR. Each individual sample was run in triplicate and has been standardized for β-actin expression levels and presented as relative expression to WT. Data (means ± SEM) are from two independent experiments with 5 mice in total per group. K Immunofluorescence showing in red Osterix (Osx)-positive cells and in blue DAPI-stained nuclei in WT and mutant mice femurs (bars: 100 μm). Dashed lines indicate the limit between the cartilage growth plate (above the line) and the bone (below the line). Images are representative of at least 3 independent determinations. L Quantification of Osx⁺ cells per mm² below the growth plate. Data (means ± SEM) are from 5, 5, and 3 independent mice in total for WT, +/1013, and 1013/1013 groups, respectively. Statistics were calculated with the unpaired two-tailed Student’s t test (1013/1013 vs WT *p = 0.0101). M Absolute numbers of the indicated stroma cell subsets from marrow fractions determined by flow cytometry. Data (means ± SEM) are from four independent experiments with n = 9, 10, and 7 mice in total for WT, +/1013, and 1013/1013 groups, respectively. Statistics were calculated with the nonparametric Kruskal–Wallis H test (^###p = 0.0004 for stroma; ^##p = 0.0013 for OPC) and the unpaired two-tailed Student’s t test (+/1013 vs WT **p = 0.0011, 1013/1013 vs WT ***p = 0.0002, +/1013 vs 1013/1013 ^§p = 0.033, for stroma; +/1013 vs WT **p = 0.0049, 1013/1013 vs WT ***p = 0.0003, for OPC). All mice were littermates, females and age-matched (8–12 wk-old). Source data are provided as a Source data file.