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Blood, Vol. 110, Issue 8, 2916-2923, October 15, 2007
Functional interplay between endothelial nitric oxide synthase and membrane type 1–matrix metalloproteinase in migrating endothelial cells
Blood Genís et al.
110: 2916
Supplemental materials for: Genis et al, Vol. 110, Issue 8, 2916-2923.
Files in this Data Supplement:
- Figure S1. Nitric oxide (NO) production was visualized by DAF-FM staining in migrating MLECs from wildtype (+/+) and eNOS null mice (−/−) (n=2) (JPG, 52.8 KB)
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No specific signal was detected in eNOS null MLECs.
- Figure S2. HUVECs were seeded at subconfluence onto DQ-COL I and left untreated (None) or treated with 100 µM DETA-NONOate or 1 µM bradykinin for 6 hours (JPG, 62.2 KB).
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DQ-COL I cleavage is visualized as fluorescent areas at cell protrusions. Collagen degradation was increased in bradykinin or DETA-NONOate-treated ECs (arrowheads). Four representative micrographs per condition are shown.
- Figure S3 MT1-MMP expression in MLECs from wildtype or eNOS null mice (JPG, 117 KB).
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(A) MT1-MMP expression was analyzed by immunofluorescence in MLECs from wildtype or eNOS null mice. Wildtype MLECs (eNOS+/+) showed concentrated MT1-MMP clusters at the membrane but eNOS−/− cells showed smaller and diffuse clusters in a point-like pattern. (B) Intensity of selected local membrane staining (white brackets) was quantified by Laserpix analysis. The histograms show the different staining profiles of eNOS+/+ and eNOS−/− MLECs. Wide peaks correspond to MT1-MMP clusters, and narrow peaks to punctate MT1-MMP staining.
- Figure S4 ) eNOS and NO production in wildtype and MT1-MMP-deficient MLECs (JPG, 112 KB).
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(A) eNOS immunofluorescence staining in wildtype and MT1-MMP-deficient MLECs. eNOS expression was similar in both cell types. (B) NO production in MLECs from MT1-MMP+/+ and MT1-MMP−/− mice. NO production, detected by DAF-FM fluorescence, showed similar localization in wildtype and MT1-MMP null cells (upper panels). Fluorescence intensity is represented in pseudocolor images to highlight the similar patterns of NO production in both cell types (lower panels).
- Figure S5. MLECs from MT1-MMP null mice were stimulated with 1 µM bradykinin for 5 minutes or 6 hours, or with EGF for 5 minutes as an activation control (JPG, 39.7 KB).
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ECs were then lysed in RIPA buffer and proteins were resolved by SDS-PAGE. ERK and phospho-ERK (p-ERK) were immunodetected by western blot. Bradykinin-induced ERK phosphorylation was detected after 5 minutes and 6 hours of stimulation.
- Video 1. NO production was visualized by DAF-FM staining in migrating HUVECs (AVI, 15 MB).
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ECs were pre-loaded with the NO probe and analyzed by real time-lapse confocal microscopy at 20 seconds interval. NO production was visualized at membrane protrusions of migrating ECs and this localized production was highly dynamic.
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