Research Paper Volume 10, Issue 6 pp 1324—1337

Integrated DNA methylation and gene expression analysis identifies SLAMF7 as a key regulator of atherosclerosis

Figure 4. High expression of SLAMF7 in CD14+ plaque monocytes stimulates proinflammatory cytokine production. (A) CD14+ macrophages were isolated from human atherosclerotic plaques, and cultured for 24 hours. SLAMF7 mRNA was detected by RT-PCR with or without LPS (10ng/ml) stimulation for 3 hours. (B) Quantification of the SLAMF7 mRNA expression in UnS vs. S plaques. P=0.03 W/O LPS, P=0.006 W LPS, by student’s t test. (C) Immunofluorescent staining of SLAMF7 in CD14+ cells from healthy donor and one UnS plaque. (D) Immunohistochemistry staining of SLAMF7 in adjacent sections with anti-SLAMF7 and CD68 antibodies, respectively. (E) Transduction with specific siRNAs targeting SLAMF7 (SLAMF7 siRNA, 200nM) in the plaque-derived CD14+ cells for 24 hours resulted in a significant suppression of protein expressions of SLAMF7, AKTser473 and PLCγ1 by Western Blot analyses. (F-G) Transduction with SLAMF7 siRNAs in the plaque-derived CD14+ cells for 24 hours resulted in a significant reduction of proinflammatory cytokines IL-6, IL-8, IL-12 and TNF-α measured by RT-PCR (F) and ELISA (G). Independent experiments were done on CD14+ monocytes from 4 different donors and the results are shown as mean ± SD. *P<0.05, vs. corresponding control, by student’s t test.