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• Figure I - (Power Point) (51 KB) oxLDL induces the CCR2 to CX3CR1 chemokine receptor switch on human monocytes. Elutriated monocytes (≈78% CD14⁺CD16^- and ≈9% CD14⁺CD16⁺) were characterized as described in Figure 1 (Results) and cultured with the indicated stimuli for 24 hours. LDL was isolated from fresh human plasma containing EDTA and sodium citrate by sequential isopycnic ultracentrifugation, washed at the upper density limit by centrifugation to remove higher density proteins, then oxidized in the presence of 20 μM CuSO₄ for 18 hours at 37 °C. The oxLDL contained 45 nmol of thiobarbituric acid-reactive substances (TBARS) per milligram of protein. The degree of LDL oxidation was also estimated by relative electrophoretic mobility i.e. electrophoretic mobility of oxLDL relative to the nonmodified native LDL summary data are shown for the % of total cells with the indicated immunophenotype as a function of cell stimulus. Data represent the mean±SD of results from 3 different donors. *P<0.05 and **P<0.01, comparing the indicated values to the corresponding unstimulated control value.
• Figure II - (Power Point) (48.5 KB) 9-HODE and 13-HODE induce monocyte production of TNFα, IFNγ and IL-1β. Human monocytes were cultured with or without 10 μg/ml of 9-HODE or 13-HODE for 24 hours. A, Analysis of receptor expression on the cell surface. Summary data are shown for the % of total cells with the indicated immunophenotype as a function of cell stimulus. B, Cell culture supernatants were analyzed for the presence of indicated cytokines by ELISA. Data represent the mean±SD from 3 independent experiments using 3 different donors with each condition tested in duplicate. *P<0.05 and **P<0.01, comparing the indicated values to the corresponding unstimulated control values.
• Figure III - (Power Point) (63 KB) PPARγ-activating glitazones promote the CCR2 to CX3CR1 chemokine receptor switch. Monocytes were cultured with the indicated stimuli for 24 hours. A through C, Analysis of receptor expression on the cell surface. Summary data are shown of the % of total cells with the indicated immunophenotype as a function of cell stimulus. Data are shown as the mean±SD from 3 independent experiments using 3 different donors. *P<0.05, comparing the indicated values to the corresponding unstimulated control values.
• Figure IV - (Power Point) (32.5 KB) The CX3CR1 promoter contains 3 consensus PPARγ response elements (PPRE). A 1278-bp region of CX3CR1 upstream of the tsp (arrow) is shown and consensus PPREs are underlined. The numbering on the left denotes the position of the first nucleotide of the corresponding line relative to the first nucleotide of the start codon. The potential PPREs and TATA box are numbered in parentheses according to the position of their 5′-most nucleotide relative to the major tsp. Sites were identified by screening with the TRANSFAC version 4.0 transcription factor data base (http://www.cbil.upenn.edu/cgi-bin/tess).
• Figure V - (Power Point) (1.1 MB) Optimization of PPARγ silencing by sRNAi in human monocytes. A, Representative experiment. Monocytes were transfected with 100 nmol/L fluorescein-labeled dsRNA (F), and the indicated concentrations of negative control or PPARγ-specific sRNAi duplexes (PPARγ1 and PPARγ2). Total RNA was isolated 24 hours after transfection and relative amounts of mRNA for the targets indicated at the lower left of each panel were examined. B, Quantitation of PPARγ results shown in A by densitometry. Data are the mean±SD from 3 independent experiments using 3 different donors. *P<0.05, comparing the indicated values to the corresponding negative control sRNAi value.
• Figure VI - (Power Point) (1.42 MB) PPARγ silencing specifically inhibits induction of monocyte PPARγ and CX3CR1 mRNA by oxidized linoleic acid metabolites. A, Cells were transfected with 200 nmol/L of the indicated oligonucleotides and cultured for 24 hours with or without 10 μg/ml of 9-HODE or 13-HODE. F indicates fluorescent oligo control. Total RNA was isolated from transfected monocytes and accumulation of mRNA for CX3CR1, γ and β-actin was examined. M, 100 bp DNA ladder. B, Quantitation of results by densitometry. Data are shown as the mean±SD from 3 independent experiments using 3 different donors. *P<0.05, comparing the indicated values to the corresponding lipid-stimulated negative control sRNAi values.