FIGURE 1.

SMPDL3A expression in cholesterol-loaded human monocyte-derived macrophages. A, SMPDL3A mRNA expression. HMDM from five individual healthy donors were incubated for 48 h in RPMI 1640 medium containing 10% (v/v) LPDS ± AcLDL (50 μg/ml), then mRNA expression measured by QRT-PCR. SMPDL3A mRNA was normalized against the housekeeping gene (β-actin) and expression in cholesterol-loaded HMDM expressed relative to nonloaded cells. Data are means ± S.D. of four technical replicates. B, free cholesterol and cholesteryl ester mass were measured using reverse phase HPLC and normalized for cell protein. Data are means ± S.D. for three determinations. C, SMPDL3A mRNA expression and comparison with SMPD1. HMDM were treated with AcLDL(50 μg/ml; 48 h), cholesterol/β-methyl cyclodextrin (Chol-CD) (20 μg/ml; 24 h), or T-090317 (T-09, 1 μm; 24 h) and then mRNA expression measured by QT-RT-PCR. SMPDL3A mRNA was normalized against the housekeeping gene (β-actin), and expression was expressed relative to untreated cells. Data are means ± S.D. of four determinations. D, SMPDL3A protein expression. HMDM cells treated as in C. Western blot analysis of SMPDL3A in cell lysates (15 μg of protein/lane) using 6E3G4A1 monoclonal antibody. Data are means ± S.D. of three determinations. Panel inset shows a representative blot. E, SMPDL3A protein secretion. HMDM¼ cells washed with serum-free media and then treated as above but using serum-free media. Conditioned media were concentrated and analyzed by anti-SMPDL3A Western blot. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (unpaired two-tailed t-tests).