Effect of leptin on lymphatic endothelial cell tube formation and proliferation.

<p>(<b>A</b>) Representative phase contrast microscopy images showing tube formation in HDLECs treated with leptin (10, 100 ng/ml) for 17 h with or without anti-leptin antibody (10 μg/ml). Scale bar: 50 μm. (<b>B</b>) Histograms represent total tube length in HDLECs exposed to leptin (10, 100 ng/ml) for 17 h with or without 1 or 10 μg/ml anti-leptin antibody. Total tube length and mean length in each condition were measured using Image J software. Results are expressed as means ± SEM of four independent experiments; *<i>p</i> < 0.05, **<i>p</i> < 0.01, <i>t</i>-test. n.s.: not significant. (<b>C</b>) WST-8 assay showing proliferation of HDLECs treated with leptin (10, 100 ng/ml) for 17 h with or without 1 or 10 μg/ml anti-leptin antibody. Histograms represent absorbance at 450 nm. Results are expressed as means ± SEM of four independent experiments; *<i>p</i> < 0.05, <i>t</i>-test. n.s.: not significant. (<b>A-C</b>) Anti-leptin antibody was added 30 min prior to leptin treatment. Ten μg/ml mouse IgG was used as an isotype control to anti-leptin antibody. (<b>D</b>) Immunoblot analysis showing the expression of claudin-5, occludin, VE-cadherin, and ZO-1 in HDLECs treated with leptin (10, 100 ng/ml) for 24 h. β-actin was used as a loading control. Histograms represent relative expression of claudin-5, occludin, VE-cadherin, and ZO-1 proteins in HDLECs exposed to 10 or 100 ng/ml leptin for 24 h as determined by densitometry analysis. Results are expressed as means ± SEM of four independent experiments; <i>t</i>-test. n.s.: not significant.</p>