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Blood, Vol. 109, Issue 10, 4264-4271, May 15, 2007
C-KIT, by interacting with the membrane-bound ligand, recruits endothelial progenitor cells to inflamed endothelium
Blood Dentelli et al.
109: 4264
Supplemental materials for: Dentelli et al, Vol 109, Issue 10, 4264-4271
Files in this Data Supplement:
- Figure S1. Kinetic analysis of mbKitL, E-selectin, and ICAM 1 expression and of EPC adhesion (JPG, 48.3 KB)
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(A) CDC-HMEC-1 cells treated with LPS, IL-1 , or TNF were analyzed for mbKitL ICAM1 or E-selectin expression by RQ-PCR at different time intervals. Densitometric quantification of mbKitL ICAM 1 or E-selectin expression is reported. Data are in arbitrary units expressed as mean ± SD. (B) Time course of EPC adhesion to TNF-treated CDC-HMEC-1 cells in the absence or in the presence of K44 or K45 is reported. Three different experiments were performed with similar results.
- Figure S2. After cytokine deprivation, CDC-HMEC-1 cells fail to release soluble KitL (JPG, 50.1 KB)
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(A) Supernatants from CDC-HMEC-1 cells untreated or treated as indicated were collected and processed by ELISA according to the manufacturer’s instruction. Data represent the mean ± SD of 3 different experiments, each performed in triplicate (*P < .05, control vs experimental groups). (B) FACS analysis was performed to analyze the expression of the mbKitL on CDC-HMEC-1 cells untreated (solid line) or treated with IL1 (bold line), TNF (dotted line), or LPS (gray line). (C) CDC-HMEC-1 cell culture media were collected upon TNF stimulation (time 0) and after deprivation, as indicated, and processed by ELISA. Data represent the mean ± SD of 4 different experiments, each performed in triplicate (*P < .05, control vs experimental groups). (D) CDC-HMEC-1 cells stimulated with TNF were deprived and analyzed for mbKitL expression by FACS analysis (left panel corresponds to time 0; right panel corresponds to 4-hour deprivation). Three different experiments were performed with similar results.
- Figure S3. EPCs depleted of endogenous c-Kit are unable to activate Akt (JPG, 30.5 KB)
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Cell extracts from EPCs transfected with c-Kit siRNA (K1) or with the scrambled sequence (scramble) were let to adhere to fibroblasts for different time intervals and subjected to SDS-PAGE. The filters were IB with anti–p–c-Kit, anti–c-Kit, anti–p-Akt, and anti-Akt antibodies, as indicated. M07e cells were used as positive control. Three different experiments were performed with similar results.
- Figure S4. Depletion of endogenous Akt in fibroblasts does not affect EPC adhesion (JPG, 28.4 KB)
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Fibroblasts transfected with an Akt siRNA or with the scrambled sequence (scramble) were lysed and subjected to 8% SDS-PAGE. The filter was IB with an anti-Akt antibody and reblotted with an anti–-actin antibody (left panel). Adhesion assay was then performed on fibroblasts depleted or not (scramble) of endogenous Akt (right panel). Adherent EPCs were counted for statistical analysis (data are the mean ± SD). Four different experiments were performed with similar results.
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