Transcriptomics reveal distinct expression signatures between the CXCR4^hi and CXCR4^lo Ly6C^hi monocyte populations. (A–E) BM cMoP, CXCR4^hi, and CXCR4^lo Ly6C^hi subsets and Ly6C^lo monocytes were sorted from three individual mice according to the gating strategy (A), and then RNA extracted for NGS. (B) PCA of gene expression. (C) Volcano plots depicting the log₂ fold change between cMoP and CXCR4^hi subset (top) and CXCR4^hi and CXCR4^lo subset (bottom) versus the −log₁₀ FDR. (D) Biofunction enrichment analysis of the top gene-ontology processes that are enriched when comparing cMoP versus CXCR4^hi subset (top) and CXCR4^hi versus CXCR4^lo subset (bottom) using IPA. (E) Heat maps of gene expression (log₂ CPM; top) and surface markers validated by flow cytometry (log₂ MFI; bottom). Flow cytometry data are representative of one out of three experiments. (F) Frequency of proliferating cells among BM monocyte subsets determined by Fucci-474 mice in vivo. Numbers in histograms (top) represent percentage of proliferating cells. Results (bottom) are expressed as mean ± SD (n = 3) and representative of two independent experiments. ****, P < 0.0001 (one-way ANOVA). (G) Mouse monocyte subsets in cell cycle stages assessed through BrdU incorporation in vivo. Results are expressed as mean ± SD (n = 5) and representative of two independent experiments. ****, P < 0.0001 (one-way ANOVA). (H) Human BM cells were pulsed with 10 µM of BrdU in vitro and monocytes in the S phase (left) were found to be CXCR4^hi, CD11b^lo, CD14^lo, and HLA-DR^hi (right). Results are representative of one out of four independent donors. (I) BM mouse monocytes subsets were assessed for their uptake of microspheres in vivo after 4 h through flow cytometry. Results are expressed as mean ± SD (n = 5) and representative of two independent experiments. **, P < 0.01 (one-way ANOVA).