Secreted HNP1-3 mediate inhibition of M-CSF–induced differentiation. (A-C) CD14⁺/CD24⁻ CMML cells (2 × 10⁵) were cultured for 4 days with M-CSF in the lower compartment of a transwell device, in the absence or presence of indicated numbers of CD14⁻/CD24⁺ CMML cells. (A) Phase-contrast microscopy analysis of CD14⁺/CD24⁻ cells. (B) RQ-PCR analysis of CD71 and CD163 mRNA in cells of the lower compartment (mean ± SD of triplicates, relative to L32 mRNA level). (C) Flow cytometric analysis of CD71 and CD163 at the surface of cells cultured in the lower compartment. Numbers indicate percentage of positive cells. Results show 1 representative of 3 independent experiments. (D-F) The supernatant of CD14⁻/CD24⁺ CMML cells cultured in serum-free medium for 4 hours was collected. HNP1-3 were depleted using clone 21 antibody coupled to beads. CD14⁺/CD24⁻ CMML cells were cultured with M-CSF for 2 days in the supernatant (depleted, not depleted, or depleted and supplemented with the 3 rhHNPs; total concentration: 5μM), before assessing CD71 and MCP1 gene expression (D; expressed as in panel B) and CD71 protein expression by immunoblot (E) or by flow cytometry after 5 days (numbers indicate % of positive cells; F).