|
|
Blood, Vol. 112, Issue 13, 4895-4904, December 15, 2008
Interleukin-1–mediated hematopoietic cell regulation in the aorta-gonad-mesonephros region of the mouse embryo
Blood Orelio et al.
112: 4895
Supplemental materials for: Orelio et al
Files in this Data Supplement:
- Table S1. RT-PCR primer sequences (PDF, 65 KB)
- Figure S1. Control optimization experiments examining the effects of IL-1β stimulation on 3T3 fibroblast cells (JPG, 48.8 KB)
-
(A) 3T3 fibroblasts were stimulated with IL-1β (10 ng/ml) for 0 min, 30 min or 2 hrs and RNA was isolated and used for cDNA synthesis. Serial dilutions of the cDNA were used for semi-quantitative RT-PCR and analysis was performed with Typhoon and ImageQuant software. IL-1β stimulation of 3T3 fibroblasts results in increased expression of the IL-1β target gene Junb as determined by semi-quantitative RT-PCR. (B) Fold induction of Junb expression induced by IL-1β stimulation in 3T3 cells. Results calculated from RT-PCR values. (C) Schematic presentation of the RT-PCR results (fold induction) show that IL-1β induced Junb gene induction in 3T3 fibroblasts can be blocked with an IL-1β blocking antibody (Ab) and that cyclohexamide (CHX) does not interfere with the induction of the immediate early Junb target gene. Cells were pre-treated with cyclohexamide (Sigma) at a final concentration of 10µg/ml for 1 hr prior to IL-1β stimulation. For blocking IL-1β, 2000 fold excess (200ng) IL-1 blocking antibody (R&D systems) was incubated with IL-1β (100 pg) for 2 hours on ice prior to addition to the cells.
- Figure S2. Engraftment of transplanted Il1r1−/− E11 AGM cells is multilineage (JPG, 40.9 KB)
-
Representative semi-quantitative PCR analysis at four months post-transplantation for (A) multilineage hematopoietic repopulation of a recipient transplanted with Il1rI−/− E11 AGM explant cells and (B) peripheral blood DNA of six secondary transplantation recipients that received bone marrow from (A). Percentage donor marker contributions (Neo gene of the Il1rI−/− locus) are indicated below each lane. Controls include the Myo gene DNA normalization and quantatitation standards containing 100, 60, 30, 10, 6, 1 and 0% donor (Neo) DNA. BL=blood; BM=bone marrow; E=erythroid; L=lymphoid; M=myeloid; Spl-spleen; T=T lymphocyte; B=B lymphocyte; Thy=thymus; LN=lymph node.
|
|
