UDP/UTP competes with HNP1-3 to inhibit monocyte differentiation. CD14⁺/CD24⁻ CMML cells were cultured in the lower compartment of a transwell device as described in Figure 5. (A) Indicated concentrations of suramin were added to the lower compartment before measuring CD71 and CD163 mRNA levels (expressed as in Figure 5B). (B) Monocytes were transfected with P2Y6 oligonucleotide sense (S) or antisense (AS) by Amaxa, 15 hours before RQ-PCR analysis of P2Y6 mRNA. Monocytes were then exposed for 4 days to rhM-CSF after indicated nucleotide transfection. Macrophage differentiation was assessed by CD71 flow cytometric analysis (P2Y6 S and P2Y6 AS are indicated by white and gray histograms, respectively; C) and morphologically (fibroblastic-like shape; D). RQ-PCR analysis of indicated gene expression in P2Y6 oligonucleotide-transfected monocytes (E). (F-G) Study of P2Y6 expression at the mRNA (F) and protein level (G) during macrophagic differentiation. HSC70 represents loading control. Molecular weights are indicated. (H-I) Primary monocytes were incubated for 3 days with rhM-CSF alone, rhM-CSF, and 5μM HNP3 in presence or absence of 100μM UTP or UDP. Macrophage differentiation was identified by increased MCP1 mRNA (H) and CD163 protein (I) expression. One of 4 independent experiments is shown.