Patino et al. 10.1073/pnas.0408032102. |
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.
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.
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.
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.