Figure 4

P₂Y₁₂ receptor mRNA is expressed in differentiated THP-1 macrophages but not naive THP-1 monocytic cells and P₂Y₁₂ receptor antigen is present on CD68 positive macrophages in human atherosclerotic plaque. 1% agarose gel showing RT-PCR results using primers specific for the P₂Y₁₂ receptor as described in the “Methods” section. Depicted gels have been cropped and the DNA standard is shown to the left of the gel (1000 bp). Uncropped gels can be found in Supplementary Figures S3, 4. (a) Monocytic cell lines (THP-1 and U937) did not express P₂Y₁₂ receptor. P₂Y₁₂ receptor mRNA expression could be detected after pre-incubation of THP-1 monocytes with washed platelets (band “THP- 1 + platelets”). (b) Once differentiated to macrophages, THP-1-derived macrophages expressed P₂Y₁₂ receptor mRNA at increasing levels 24, 48 and 72 h after induction of macrophage differentiation. (c) Anti-CD68 and anti-P₂Y₁₂ immunofluorescence on human carotid atherosclerotic plaques obtained by carotid endarterectomy. Preparation and staining were performed as described in the “Methods” section. The different channels are named at the top of the images (“Merged”, “DAPI” etc.). Immunofluorescence revealed that CD68⁺ macrophages within the atherosclerotic plaque also expressed the P₂Y₁₂ receptor. Scale bar = 50 µm. MC = CD68⁺ macrophage concentrated area.