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Figure 5 | BMC Complementary and Alternative Medicine

Figure 5

From: Plant proteolytic enzyme papain abrogates angiogenic activation of human umbilical vein endothelial cells (HUVEC) in vitro

Figure 5

Effect of proteolytic enzymes on cell migration and tube formation in HUVEC. HUVEC were seeded into fibronectin coated 12-well microtiterplates and cultured in EGM. After 24 hours medium was changed to EBM. Cells were stained using calcein-AM, the monolayer was scratched with a 200 μL pipette tip and photographed at 10 fold magnification using a Nikon Eclipse Ti as described above. After 14 hours culture in EBM containing 10 ng/mL VEGF or 10 ng/mL VEGF and papain and photographed. covered area was measured using the TScratch software package, results were calculated as percent open area. Data are shown as mean ± SEM. Panel A shows photomicrographs at the beginning of the assay (column start) and after 14 hours incubation (column end) for cells treated with either 10 ng/mL VEGF or 10 ng/mL VEGF and 10 μg/mL papain. Migration fronts are marked by white lines. Panel B shows the percentage open area. Papain inhibited cell migration almost completely at a concentration of 10 μg/mL. Angiogenesis slides were coated with 10 μL Matrigel per well and incubated for 30 min at 37°C. Endothelial cells were and seeded into the wells at a density of 5000 cells per well in EBM containing 10 ng/mL VEGF. After 4 hours preincubation, papain was added at concentrations as indicated. After a further 20 hour incubation period, cells were labelled with 2 μM Calcein-AM. Micrographs of fluorescent cells were taken at 4 fold magnification using a Nikon Eclipse Ti as described above. Tube formation was quantified by the angiogenesis analyzer plugin for ImageJ. Panel C shows photomicrographs for the control, treatment with 10 ng/mL VEGF and with 10 ng/mL VEGF in combination with 1 and 10 μg/mL papain. Panel D shows the measured network length as percentage untreated control. Tube length decreased significantly to control levels after treatment with 1 μg/mL papain. At concentrations of 10 μg/mL tube formation was almost completely abrogated.

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