Patino et al. 10.1073/pnas.0408032102.

Supporting Information

Files in this Data Supplement:

Supporting Figure 4
Supporting Table 3
Supporting Table 4
Supporting Table 5
Supporting Figure 5




Supporting Figure 4

Fig. 4. Confirmation of monocyte and macrophage purity. (A) Flow cytometry showing the relative distribution profiles of CD14- (negative) and CD14+ (positive with anti-CD14 antibody conjugated to FITC) cells in the mononuclear cell (MNC), purified monocyte (Mono), and monocyte-depleted (Non-mono) fractions. (B) RT-PCR of undiluted (labeled 1) and one-tenth diluted (0.1) cDNA from various cellular fractions of blood and carotid plaque tissue. The cell-specific markers used were as follows: control genes, glyceraldehyde-3-phosphate dehydrogenase (GAPD) and translation initiation factor (TIF); monocyte and macrophage, CD14; macrophage, macrophage mannose receptor (CD206); lymphocyte, CD3; platelet, glycoprotein IIb (GPIIb). NTC, no template control; RT-, no reverse transcriptase enzyme control; SN, plaque supernatant cells after CD14+ macrophage depletion. For monocyte purity, note the absence of CD3 and GpIIb in the Mono fraction. After macrophage purification from the Total plaque suspension, note the depletion of the macrophage marker CD206 in the remaining tissue SN fraction.





Supporting Figure 5

Fig. 5. Finkel-Biskis-Jinkins osteosarcoma (FOS) and dual specificity phosphatase 1 (DUSP1) relative mRNA expression levels determined by RT-PCR. The fold change ratios in patients compared with controls [Ratio (P/C)] were preserved whether whole mononuclear cells (MNC) or purified monocytes (Monocyte) were used for RT-PCR. Values shown as mean ± SE, n = 6, for patients and controls.





 

Table 3. RT-PCR primer sequences

Gene

Forward (5' - 3')

Reverse (5' - 3')

Human

GAPD

CATCTCTGCCCCCTCTGCT

ACGCCTGCTTCACCACCTT

TIF

GACACAAGTCTCCAGAACGGC

TGGTCTCAAAGTCATCGGGAA

FOS

GGAGGACCTTATCTGTGCGTGA

GAACACACTATTGCCAGGAACACA

DUSP1

GGAGGACAACCACAAGGCAGA

TGTGTCGTCGGGAATAATACTGGT

NFKBIA

TACGAGCAGATGGTCAAGGAGC

TTCAGGATGGAGTGGAGGTGC

ID2

CCCAGAACAAGAAGGTGAGCAA

CAAGTAAGAGAACACCCTGGGAAG

PER1

TCCAGTCCAGCCTTACCTACAGC

CCAACCCTCAAGAGTCAGATTCAG

SAP30

GCATCTCCCAGAAGAAGGTGAAG

TAAGTCCTGGTCTGGTTGGTAGC

CD14

TCCGAAGCCTTCCAGTGTGT

ACAGAGAGCCGCCATCAGTC

CD206

TGGTTTCCATTGAAAGTGCTGC

TTCCTGGGCTTGACTGACTGTTA

CD3

TTCCCAACCCAGACTATGAGC

AAGGAGGGAACTGAACGGAG

GPIIb

ACAGATCTTCCTGCCAGAGC

CACCCACCAGATTGGAATGGC

Primer pair sequences used for quantitative RT-PCR of the various listed genes. Full gene names appear in the corresponding figure legends where the primer pairs were utilized.





Supporting Table 4

Table 4. List of 297 genes increased in monocytes of atherosclerosis patients. Mon, monocytes; nonmon, monocyte-depleted mononuclear cells. Subjects with atherosclerosis: P1, patient 1; P2, patient 2. Subjects without clinically significant atherosclerosis: C1, age-matched control 1; A1, younger age control 1; A2, younger age control 2.





Supporting Table 5

Table 5. List of 267 genes decreased in monocytes of atherosclerosis patients. Mon, monocytes; nonmon, monocyte-depleted mononuclear cells. Subjects with atherosclerosis: P1, patient 1; P2, patient 2. Subjects without clinically significant atherosclerosis: C1, age-matched control 1; A1, younger age control 1; A2, younger age control 2.