Journal: PLoS ONE

Article Title: Novel Mechanisms of Compromised Lymphatic Endothelial Cell Homeostasis in Obesity: The Role of Leptin in Lymphatic Endothelial Cell Tube Formation and Proliferation

doi: 10.1371/journal.pone.0158408

Figure Lengend Snippet: ( A ) Phase contrast microscopy image showing the morphology of HDLECs used in this study. Scale bar: 1 μm. ( B ) Real-time PCR to confirm the expression of VEGFR-3 mRNA in human HDLECs and HUVECs. Histograms represent the relative expression of VEGFR-3 mRNA. Results are expressed as means ± SEM of three independent experiments; ***p < 0.001, t -test. PCR ( C ) and immunoblot analysis ( D ) to confirm the expression of leptin receptor (Ob-R) mRNA ( C ) and protein ( D ) in HDLECs and HUVECs. ( B and C ) GAPDH was used as an internal control. bp: base pair. ( D ) β-actin was used as a loading control.

Article Snippet: The following primary monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs) were used to detect proteins by immunofluorescence labeling and immunoblot analysis and/or to neutralize the effects of leptin and IL-6: mouse anti-leptin mAb (44802), anti-leptin receptor mAb (52263) (R&D Systems, Minneapolis, MN, USA), rat anti-IL-6 mAb (MQ2-13A5; Miltenyi Biotec, Bergisch Gladbach, Germany), mouse anti-phospho STAT3 (Tyr705) mAb (3E2), rabbit anti-STAT3 pAb (D47E7), anti-SOCS3 pAb (Cell Signaling Technology, Danvers, MA, USA), mouse anti-β-actin mAb (E1C605; EnoGene, New York, NY, USA), rabbit anti-claudin-5 mAb (EPR7583), anti-VE-cadherin pAb (Abcam, Cambridge, MA, USA), rabbit anti-occludin pAb, anti-ZO-1 pAb (Invitrogen, Carlsbad, CA, USA), and mouse anti-D2-40 mAb (413451; Nichirei, Tokyo, Japan).

Techniques: Microscopy, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Control

Journal: PLoS ONE

Article Title: Novel Mechanisms of Compromised Lymphatic Endothelial Cell Homeostasis in Obesity: The Role of Leptin in Lymphatic Endothelial Cell Tube Formation and Proliferation

doi: 10.1371/journal.pone.0158408

Figure Lengend Snippet: ( A ) 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 ) 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; * p < 0.05, ** p < 0.01, t -test. n.s.: not significant. ( C ) 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; * p < 0.05, t -test. n.s.: not significant. ( A-C ) 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. ( D ) 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; t -test. n.s.: not significant.

Article Snippet: The following primary monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs) were used to detect proteins by immunofluorescence labeling and immunoblot analysis and/or to neutralize the effects of leptin and IL-6: mouse anti-leptin mAb (44802), anti-leptin receptor mAb (52263) (R&D Systems, Minneapolis, MN, USA), rat anti-IL-6 mAb (MQ2-13A5; Miltenyi Biotec, Bergisch Gladbach, Germany), mouse anti-phospho STAT3 (Tyr705) mAb (3E2), rabbit anti-STAT3 pAb (D47E7), anti-SOCS3 pAb (Cell Signaling Technology, Danvers, MA, USA), mouse anti-β-actin mAb (E1C605; EnoGene, New York, NY, USA), rabbit anti-claudin-5 mAb (EPR7583), anti-VE-cadherin pAb (Abcam, Cambridge, MA, USA), rabbit anti-occludin pAb, anti-ZO-1 pAb (Invitrogen, Carlsbad, CA, USA), and mouse anti-D2-40 mAb (413451; Nichirei, Tokyo, Japan).

Techniques: Microscopy, Software, Control, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Novel Mechanisms of Compromised Lymphatic Endothelial Cell Homeostasis in Obesity: The Role of Leptin in Lymphatic Endothelial Cell Tube Formation and Proliferation

doi: 10.1371/journal.pone.0158408

Figure Lengend Snippet: Immunoblot analysis of pSTAT3 (Y705), STAT3 and SOCS3 in HDLECs exposed to 10 or 100 ng/ml leptin for 15 min ( A ) and 24 h ( B ). β-actin was used as a loading control. Histograms represent relative expression of pSTAT3 and SOCS3 proteins in HDLECs exposed to 10 or 100 ng/ml leptin for 15 min ( A ) and 24 h ( B ) as determined by densitometry analysis. Results are expressed as means ± SEM of four independent experiments; * p < 0.05, ** p < 0.01, *** p < 0.001, t -test. n.s.: not significant. ( C ) Histograms represent total tube length in HDLECs exposed to leptin (100 ng/ml) for 17 h with or without 250 nM STAT3 inhibitor. 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; ** p < 0.01, t -test. n.s.: not significant. ( D ) WST-8 assay showing proliferation of HDLECs treated with leptin (100 ng/ml) for 17 h with or without 250 nM STAT3 inhibitor. Histograms represent absorbance at 450 nm. Results are expressed as means ± SEM of four independent experiments; * p < 0.05, t -test. n.s.: not significant. ( C and D ) STAT3 inhibitor was added 30 min prior to leptin treatment.

Article Snippet: The following primary monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs) were used to detect proteins by immunofluorescence labeling and immunoblot analysis and/or to neutralize the effects of leptin and IL-6: mouse anti-leptin mAb (44802), anti-leptin receptor mAb (52263) (R&D Systems, Minneapolis, MN, USA), rat anti-IL-6 mAb (MQ2-13A5; Miltenyi Biotec, Bergisch Gladbach, Germany), mouse anti-phospho STAT3 (Tyr705) mAb (3E2), rabbit anti-STAT3 pAb (D47E7), anti-SOCS3 pAb (Cell Signaling Technology, Danvers, MA, USA), mouse anti-β-actin mAb (E1C605; EnoGene, New York, NY, USA), rabbit anti-claudin-5 mAb (EPR7583), anti-VE-cadherin pAb (Abcam, Cambridge, MA, USA), rabbit anti-occludin pAb, anti-ZO-1 pAb (Invitrogen, Carlsbad, CA, USA), and mouse anti-D2-40 mAb (413451; Nichirei, Tokyo, Japan).

Techniques: Western Blot, Control, Expressing, Software

Journal: PLoS ONE

Article Title: Novel Mechanisms of Compromised Lymphatic Endothelial Cell Homeostasis in Obesity: The Role of Leptin in Lymphatic Endothelial Cell Tube Formation and Proliferation

doi: 10.1371/journal.pone.0158408

Figure Lengend Snippet: Immunoblot analysis of pSTAT3 (Y705), STAT3 and SOCS3 in HDLECs exposed to 10 or 100 ng/ml leptin for 15 min ( A ) and 24 h ( B ). β-actin was used as a loading control. Histograms represent relative expression of pSTAT3 and SOCS3 proteins in HDLECs exposed to 10 or 100 ng/ml leptin for 15 min ( A ) and 24 h ( B ) as determined by densitometry analysis. Results are expressed as means ± SEM of four independent experiments; * p < 0.05, ** p < 0.01, *** p < 0.001, t -test. n.s.: not significant. ( C ) Histograms represent total tube length in HDLECs exposed to leptin (100 ng/ml) for 17 h with or without 250 nM STAT3 inhibitor. 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; ** p < 0.01, t -test. n.s.: not significant. ( D ) WST-8 assay showing proliferation of HDLECs treated with leptin (100 ng/ml) for 17 h with or without 250 nM STAT3 inhibitor. Histograms represent absorbance at 450 nm. Results are expressed as means ± SEM of four independent experiments; * p < 0.05, t -test. n.s.: not significant. ( C and D ) STAT3 inhibitor was added 30 min prior to leptin treatment.

Article Snippet: The following primary monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs) were used to detect proteins by immunofluorescence labeling and immunoblot analysis and/or to neutralize the effects of leptin and IL-6: mouse anti-leptin mAb (44802), anti-leptin receptor mAb (52263) (R&D Systems, Minneapolis, MN, USA), rat anti-IL-6 mAb (MQ2-13A5; Miltenyi Biotec, Bergisch Gladbach, Germany), mouse anti-phospho STAT3 (Tyr705) mAb (3E2), rabbit anti-STAT3 pAb (D47E7), anti-SOCS3 pAb (Cell Signaling Technology, Danvers, MA, USA), mouse anti-β-actin mAb (E1C605; EnoGene, New York, NY, USA), rabbit anti-claudin-5 mAb (EPR7583), anti-VE-cadherin pAb (Abcam, Cambridge, MA, USA), rabbit anti-occludin pAb, anti-ZO-1 pAb (Invitrogen, Carlsbad, CA, USA), and mouse anti-D2-40 mAb (413451; Nichirei, Tokyo, Japan).

Techniques: Western Blot, Control, Expressing, Software

Journal: PLoS ONE

Article Title: Novel Mechanisms of Compromised Lymphatic Endothelial Cell Homeostasis in Obesity: The Role of Leptin in Lymphatic Endothelial Cell Tube Formation and Proliferation

doi: 10.1371/journal.pone.0158408

Figure Lengend Snippet: Leptin derived from adipocytes regulates tube formation through controlling proliferation of HDLECs. Although leptin activates the STAT3 signaling pathway, which is downstream signaling of Ob-R, at an early time point, STAT3 phosphorylation is not increased at a late time point when SOCS3 proteins are increased by leptin. As a result, proliferation of HDLECs is decreased, leading to inhibition of tube formation. Additionally, SOCS3 induced by leptin is under the regulation of STAT3. Leptin also enhanced expression of the proinflammatory cytokine IL-6 in HDLECs. IL-6 rescues the compromised proliferation and tube formation caused by leptin in an autocrine or paracrine manner.

Article Snippet: The following primary monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs) were used to detect proteins by immunofluorescence labeling and immunoblot analysis and/or to neutralize the effects of leptin and IL-6: mouse anti-leptin mAb (44802), anti-leptin receptor mAb (52263) (R&D Systems, Minneapolis, MN, USA), rat anti-IL-6 mAb (MQ2-13A5; Miltenyi Biotec, Bergisch Gladbach, Germany), mouse anti-phospho STAT3 (Tyr705) mAb (3E2), rabbit anti-STAT3 pAb (D47E7), anti-SOCS3 pAb (Cell Signaling Technology, Danvers, MA, USA), mouse anti-β-actin mAb (E1C605; EnoGene, New York, NY, USA), rabbit anti-claudin-5 mAb (EPR7583), anti-VE-cadherin pAb (Abcam, Cambridge, MA, USA), rabbit anti-occludin pAb, anti-ZO-1 pAb (Invitrogen, Carlsbad, CA, USA), and mouse anti-D2-40 mAb (413451; Nichirei, Tokyo, Japan).

Techniques: Derivative Assay, Phospho-proteomics, Inhibition, Expressing