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Blood, Vol. 113, Issue 2, 429-437, January 8, 2009
Serum amyloid A induces G-CSF expression and neutrophilia via Toll-like receptor 2
Blood He et al.
113: 429
Supplemental materials for: He et al
Files in this Data Supplement:
- Figure S1. SAA-induced G-CSF secretion from macrophages is dependent on TLR2 but not TLR4 (JPG, 59.2 KB)
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BMDM from wild type C57BL/6, Tlr2−∕−, and Tlr4lps-del mice were stimulated with SAA, or lipopeptides Pam3 and Pam2, or LPS for 24h, and the secreted G-CSF was determined using ELISA. Data are presented as means ± SEM of three experiments, each performed in duplicate.
- Figure S2. SAA does not synergistically enhance the effect of lipopeptide on G-CSF secretion (JPG, 27.5 KB)
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Mouse BMDM cells were stimulated with different concentrations of SAA (0.1 and 1 µM) or FSL-1 (0.01 and 0.1 µg/ml) or their mixtures for 24h. The secreted G-CSF was determined using ELISA. Data are presented as means ± SEM of three experiments, each performed in duplicate.
- Figure S3. The antagonists of FPRL1/ALX cannot inhibit the SAA-stimulated TLR2-independent G-CSF secretion (JPG, 28.7 KB)
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BMDM from wild type C57BL/6 and Tlr2−∕− mice were pretreated with or without peptides t-Boc (10 µM) or WRW4 (10 µM) for 30 min, and then stimulated with SAA (1µM) for 24h. The secreted G-CSF was measured using ELISA. Data are presented as means ± SEM of three experiments, each performed in duplicate.
- Figure S4. The expression of TLR1, but not TLR6 or CD14, increases SAA-induced G-CSF luciferase activity in HeLa/TLR2 cells (JPG, 51.2 KB)
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HeLa/TLR2 and HeLa/vector cells were transfected with G-CSF luciferase reporter construct, pCMVβ vector DNA and/or other expression constructs as indicated. The transfected cells were then stimulated with SAA (0.1 µM), or Pam3CSK4 (1 µg/ml), or PGN (1 µg/ml), or LTA (10 µg/ml), or zymosan (100 µg/ml) for 5 h. The luciferase activities were determined. Data are presented as means ± SEM of 2–4 experiments, each performed in triplicate.
- Figure S5. SAA induces NF-κB activation in the mouse BMDM (JPG, 20.1 KB)
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This is the whole gel picture of Fig. 3B.
- Figure S6. Lipoprotein lipase pretreatment does not affect SAA-induced G-CSF secretion from BMDM (JPG, 97 KB)
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(A) Different concentrations of lipoprotein lipase (0.025, 0.05, and 0.1 mg/ml) were used to pretreat SAA (1 µM), or Pam3CSK4 (1 µg/ml), or FSL-1 (1 µg/ml) for 1 h at 37°C, then heated at 100°C for 5 min. The lipase-treated SAA, Pam3CSK4, and FSL-1 were then used to stimulate mouse BMDM for 16 h. The secreted G-CSF was measured using ELISA. Short exposure of SAA, Pam3CSK4, or FSL-1 to heat (100°C for 5 min) without pretreatment with lipoprotein lipase was used as controls. (B) Mouse BMDM cells were stimulated for 16 h with the short-time heat-treated SAA, Pam3CSK4, FSL-1 or lipoprotein lipase (100°C for 5 min), and the secreted G-CSF was compared with untreated cells. Data are presented as means ± SEM of 2–4 experiments, each performed in duplicate.
- Figure S7. Lipoprotein lipase inhibits Pam3CSK4-induced G-CSF secretion from BMDM (JPG, 58.6 KB)
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Different concentrations of lipoprotein lipase (0.025, 0.05, and 0.1 mg/ml) were used to pretreat Pam3CSK4 (1 µg/ml) for 30 min at 37°C , then heated at 72°C for 30 min. The lipase-treated Pam3CSK4 was then used to stimulate mouse BMDM for 16 h at 37°C . The secreted G-CSF was measured using ELISA. Exposure of Pam3CSK4 to heat (37°C for 30 min then 72°C for 30 min) without pretreatment with lipase was used as a control. Lipase alone (72°C for 30 min) was also used to stimulate mouse BMDM cells. Data are presented as means ± SEM of 2–4 experiments, each performed in duplicate.
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