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Blood, Vol. 109, Issue 11, 4793-4802, June 1, 2007
Kringle 5 of human plasminogen, an angiogenesis inhibitor, induces both autophagy and apoptotic death in endothelial cells
Blood Nguyen et al.
109: 4793
Supplemental materials for: Nguyen et al, Vol 109, Issue 11, 4793-4802
Files in this Data Supplement:
- Figure S1. The expression of p53 in rK5-treated HUVECs (JPG, 89 KB)
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Western blot analysis of p53 in rK5-treated HUVECs. (A) Total-cell lysates from rK5-treated HUVECs were collected at different time points and subjected to Western blot analysis. (B) Densitometric analysis of changes in p53 levels is shown. Values were normalized against β-actin and presented as fold increase compared with the basal level (0 hour).
- Figure S2. Treatment with rK5 depolarized mitochondrial ΔΨM of HUVECs cultured in complete medium (JPG, 88.8 KB)
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HUVECs were cultured in complete medium (10% FBS) for 24 hours in the presence or absence of 1.5 µg/mL rK5. Subsequently, treated cells were incubated with TMRE (40 nM) for 15 minutes prior to harvesting. Representative FACS profiles are shown. Heat-inactivated rK5 was used as a control.
- Figure S3. Phase-contrast bright-field images of rK5-treated endothelial cells (JPG, 190 KB)
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HUVECs were seeded in chamber slides in M199 containing 5% FBS supplemented with or without 40 ng/mL VEGF. Recombinant K5 was added at a concentration of 3 µg/mL. Images were recorded at 400× magnification after 24 hours of treatment.
- Figure S4. Specificity of rK5-induced autophagy: confocal images of rK5-treated fibroblasts and ovarian cancer cells (JPG, 298 KB)
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Human foreskin fibroblasts and the ovarian cancer cell line MA148 were seeded in chamber slides. Cells were then treated with rK5 (3 µg/mL) for 24 hours in complete serum media (for MA148) and 5% FBS in M199 medium supplemented with VEGF (for fibroblasts). Cells were labeled with 0.05 mM MDC (red; top panel) and 1 µM Mitotracker Green. Magnification, 600×. Overlay images of MDC and Mitotracker Green are shown in the bottom panel. Etoposide (10 µg/mL) was used as a positive control.
- Figure S5. LAMP1 detection in rK5-treated HUVECs (JPG, 386 KB)
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HUVECs were cultured in reduced serum medium supplemented with 40 ng/mL VEGF. Endothelial cells were treated with purified rAAV-GFP CM or rK5 (1.5 µg/mL) for 24 hours. LAMP1 was detected using a rabbit polyclonal anti-human LAMP1 antibody (1:100) and goat anti-rabbit Alexa 488 (1:500; green). Mitochondria were visualized by Mitotracker Red. Confocal images were recorded at 600 × magnification.
- Figure S6. Beclin 1 down-regulation in HUVECs potentiates rK5-induced apoptosis: analysis of total cell population (JPG, 50 KB)
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HUVECs were transfected with either shRNA specific to Beclin 1 (white blocks) or a scrambled shRNA (black blocks) using Lipofectamine 2000. After transfection, HUVECs were cultured in 5% FBS supplemented with 40 ng/mL VEGF and then treated with 1.5 µg/mL rK5. Scrambled shRNA-transfected HUVECs (black diamonds) and shRNA to Beclin 1–transfected HUVECs (white diamonds) were used as negative controls for the TUNEL assay. (A) The percentages of caspase-activated HUVECs at different time points are shown. (B) Percentage of apoptotic HUVECs at different time points was determined by the ratio between TUNEL+ cells and the total number of cells per field. Data represent values from 2 independent experiments. Samples were run in triplicate. Values are shown as means ± SE (*P < .05; **P < .001).
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