Journal: Frontiers in Cardiovascular Medicine

Article Title: Lymphatic endothelial sphingosine 1-phosphate receptor 1 enhances macrophage clearance via lymphatic system following myocardial infarction

doi: 10.3389/fcvm.2022.872102

Figure Lengend Snippet: The expression of S1pr1 was down-regulated in cardiac lymphatic endothelial cells. (A) Relative mRNA expression levels of S1pr1 in cardiac LECs of mice at 3 days after sham or LAD operation ( n = 6). (B) Schematic diagram of generation of mice with LEC-specific loss of S1pr1 allele. (C) Relative mRNA expression levels of S1pr1 in cardiac LECs of WT and Lyve1-Cre-S1pr1 flox/wt mice ( n = 3). (D) Western-blotting analysis of S1pr1 expression in cardiac LECs from the indicated groups, with its quantification ( n = 3). Data are mean ± S.E.M.

Article Snippet: Human lymphatic endothelial cells (HLECs, ATCC) were cultivated in EGM2 (Endothelial cell growth medium 2, PromoCell) and cells within 8th passage were used for in vitro experiments.

Techniques: Expressing, Western Blot

Journal: Frontiers in Cardiovascular Medicine

Article Title: Lymphatic endothelial sphingosine 1-phosphate receptor 1 enhances macrophage clearance via lymphatic system following myocardial infarction

doi: 10.3389/fcvm.2022.872102

Figure Lengend Snippet: S1pr1 regulates lymphatic endothelial cell migration, proliferation, and angiogenic activity. (A) RT-qPCR analysis of S1PR1 mRNA levels in LECs of the indicated group ( n = 3). (B) MTT assay showed the proliferation of cells of the indicated group ( n = 3). (C) Scratch wound healing assay showed the cell migration, with quantification of cell migration area (%) of the indicated group ( n = 3). (D) Boyden chamber assay showed the cell migration of cells of the indicated group, with quantification of migrated cells of the indicated group ( n = 3). (E) Tube formation assay showed S1PR1 shRNA promoted LEC tube formation, with quantification of the number of branch points ( n = 3). Control, scramble RNA lentivirus. OE, S1PR1-overexpressing lentivirus. shRNA, S1PR1 shRNA lentivirus. Data are mean ± S.E.M. Scale Bars: ( C–E) , 100 μm.

Article Snippet: Human lymphatic endothelial cells (HLECs, ATCC) were cultivated in EGM2 (Endothelial cell growth medium 2, PromoCell) and cells within 8th passage were used for in vitro experiments.

Techniques: Migration, Activity Assay, Quantitative RT-PCR, MTT Assay, Wound Healing Assay, Boyden Chamber Assay, Tube Formation Assay, shRNA, Control

Journal: Frontiers in Cardiovascular Medicine

Article Title: Lymphatic endothelial sphingosine 1-phosphate receptor 1 enhances macrophage clearance via lymphatic system following myocardial infarction

doi: 10.3389/fcvm.2022.872102

Figure Lengend Snippet: Working model of lymphatic endothelial S1pr1 enhances macrophage clearance via lymphatic system following myocardial infarction.

Article Snippet: Human lymphatic endothelial cells (HLECs, ATCC) were cultivated in EGM2 (Endothelial cell growth medium 2, PromoCell) and cells within 8th passage were used for in vitro experiments.

Techniques:

Journal: Frontiers in Oncology

Article Title: circUBE2G1 interacts with hnRNPU to promote VEGF-C-mediated lymph node metastasis of lung adenocarcinoma

doi: 10.3389/fonc.2024.1455909

Figure Lengend Snippet: CircUBE2G1 promotes lymphangiogenesis in vitro . (A, B) Representative images and quantification of tube formation and migration of HLECs treated with culture medium from A549 (A) or H1975 (B) cells with circUBE2G1 knockdown or overexpression. Scale bars, 100 μm. Statistical significances between two groups were assessed using two-tailed t-test, and statistical significances between multiple groups was assessed using one-way ANOVA followed by Dunnett’s t-test. Error bars represent the standard deviation of three independent experiments. *p < 0.05, **p < 0.01 .

Article Snippet: Human lymphatic endothelial cells (HLECs) were purchased from ScienCell Research Laboratories (Carlsbad, California, USA, Cat# 2500) and cultured in endothelial cell medium (ECM; ScienCell Research Laboratories, Cat# 1001) supplemented with 5% FBS.

Techniques: In Vitro, Migration, Knockdown, Over Expression, Two Tailed Test, Standard Deviation

Journal: Frontiers in Oncology

Article Title: circUBE2G1 interacts with hnRNPU to promote VEGF-C-mediated lymph node metastasis of lung adenocarcinoma

doi: 10.3389/fonc.2024.1455909

Figure Lengend Snippet: CircUBE2G1 promotes lymphangiogenesis and LN metastasis in LUAD by enhancing VEGF-C expression. (A, B) Representative images and quantification of tube formation and migration of HLECs induced by conditioned medium from A549 (A) or H1975 (B) cells under different treatments. Scale bars, 100 μm. (C) Representative images of anti-mCherry immunohistochemical staining of popliteal LNs in nude mice ( n = 12 per group). Red scale bars, 500 μm; black scale bars, 100 μm. (D) Popliteal LN metastasis rate across all groups ( n = 12 per group). (E) Kaplan-Meier survival curves ( n = 12 per group). Statistical differences were evaluated using the two-tailed student t-test in (A, B) and the χ2 test in (D) . Error bars represent the standard deviation for three independent experiments. *P < 0.05; **P < 0.01 .

Article Snippet: Human lymphatic endothelial cells (HLECs) were purchased from ScienCell Research Laboratories (Carlsbad, California, USA, Cat# 2500) and cultured in endothelial cell medium (ECM; ScienCell Research Laboratories, Cat# 1001) supplemented with 5% FBS.

Techniques: Expressing, Migration, Immunohistochemical staining, Staining, Two Tailed Test, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Extracellular Hsp90α Promotes Tumor Lymphangiogenesis and Lymph Node Metastasis in Breast Cancer

doi: 10.3390/ijms22147747

Figure Lengend Snippet: eHsp90α enhances migration and tube formation abilities of LECs. ( A ) Quantification results of the cell viability of hLECs treated with recombinant Hsp90α protein at different dosages in the culture medium containing 1% FBS. Culture medium with 10% FBS was used as the positive control. ( B , C ) Representative images and quantification results of the cell migration ability ( B ) and the tube formation ability ( C ) of hLECs treated with indicated reagents (200 ng/mL Hsp90α or control BSA for the tube formation assay). Scale bar, 200 μm. ( D , E ) Representative images and quantification results of the cell migration ability ( D ) and the tube formation ability ( E ) of hLECs in the presence of MDA-MB-231 CM, which was pre-mixed with control IgG or Hsp90α neutralizing antibody (200 ng/mL) for 30 min at 37 °C. Scale bar, 200 μm. ( F , G ) Matrigel plugs containing PBS, BSA or recombinant Hsp90α protein ( F ) and plugs containing MDA-MB-231 CM, which was pre-incubated with control IgG or Hsp90α neutralizing antibody ( G ) were subcutaneously injected into BALB/c mice. After 8 days, plugs were applied to IF analysis. Representative images of lymphatic vessel (green staining of LYVE-1) were displayed (top). Quantification results were shown (bottom). Concentration of recombinant Hsp90α protein or Hsp90α neutralizing antibody is 1 μg/mL. Scale bar, 100 μm. Data from three independent experiments are represented as mean ± SD. ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Human dermal lymphatic endothelial cells (hLECs) were purchased from ScienCell Research Laboratories and cultured under the manufacturer’s instructions.

Techniques: Migration, Recombinant, Positive Control, Control, Tube Formation Assay, Incubation, Injection, Staining, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Extracellular Hsp90α Promotes Tumor Lymphangiogenesis and Lymph Node Metastasis in Breast Cancer

doi: 10.3390/ijms22147747

Figure Lengend Snippet: eHsp90α mediates lymphangiogenic activities through the LRP1-AKT pathway. ( A ) Detection of LRP1 (red staining) in the newly formed lymphatic vessels (indicated by LYVE-1, green staining) from the Matrigel plug assay by IF. Scale bar, 50 μm. ( B , C ) Representative images and quantification results of the migration ability ( B ) and tube formation ability ( C ) of hLECs induced by Hsp90α (200 ng/mL) when cells were pretreated with LRP1 neutralizing antibody (200 ng/mL). Scale bar, 200 μm. ( D ) Matrigel plugs containing BSA or Hsp90α protein (1 μg/mL) in the presence of lentivirus-delivered Lrp1 shRNA or negative control (2.5 × 10 6 U) were subcutaneously injected into BALB/c mice. After 8 days, plugs were applied to IF analysis. Lymphatic vessel (green staining of LYVE-1) was displayed (left). Quantification results were shown (right). Scale bar, 100 μm. ( E ) hLECs were stimulated by recombinant Hsp90α protein in the presence of LRP1 neutralizing antibody or its isotype IgG control. The protein levels of p-AKT, p-ERK, AKT and ERK were measured by Western blotting. Quantitative results are shown below. ( F ) The protein levels of p-AKT and AKT in hLECs exposed to recombinant Hsp90α protein at indicated points of time were determined by Western blotting (left). The protein levels of p-AKT and AKT in hLECs treated with indicated regeants were detected by Western blot analysis (right). Quantitative results are shown below. ( G, H ) hLECs were pretreated with MK-2206 (inhibitor of AKT, 1 μM) or DMSO for 30 min. Representative images and quantification results of the cell migration ability ( G ) and tube formation ability ( H ) of inhibitor-pretreated hLECs induced by recombinant Hsp90α protein. Scale bar, 200 μm. Data are represented as mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Human dermal lymphatic endothelial cells (hLECs) were purchased from ScienCell Research Laboratories and cultured under the manufacturer’s instructions.

Techniques: Staining, Matrigel Assay, Migration, shRNA, Negative Control, Injection, Recombinant, Control, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Extracellular Hsp90α Promotes Tumor Lymphangiogenesis and Lymph Node Metastasis in Breast Cancer

doi: 10.3390/ijms22147747

Figure Lengend Snippet: CXCL8 functions in the eHsp90α-induced lymphangiogenic process in hLECs. ( A ) The transcriptome levels of hLECs in the Hsp90α Ab group and the IgG control group were analyzed. Volcano plot was drawn to present the upregulated and downregulated DEGs by Hsp90α neutralizing antibody treatment. In total, 59 upregulated DEGs and 118 downregulated DEGs were colored in red and blue, respectively. ( B ) Venn diagram was drawn to show the overlapping genes between upregulated DEGs and negative-related lymphangiogenic genes, also between downregulated DEGs and positive-related lymphangiogenic genes. ( C ) Validation of RNA-seq results by qRT-PCR. ( D ) Further exploration of CCL24, CXCL8, HDAC9 expression in hLECs treated with recombinant Hsp90α protein by qRT-PCR. ( E ) Immunoblotting analyses of CXCL8 expression stimulated by recombinant Hsp90α protein in the presence of Hsp90α neutralizing antibody or control IgG. Quantitative results are shown below. ( F ) The protein level of CXCL8 induced by recombinant Hsp90α protein with or without MK-2206. Quantitative results are shown below. ( G ) Quantification of secreted CXCL8 levels in hLEC supernatant measured by ELISA. ( H , I ) Representative and quantified data showing the relative migration ( H ) and tube formation ( I ) activities of hLECs stimulated with recombinant Hsp90α protein (200 ng/mL) or CXCL8 protein (1 ng/mL) in the presence or absence of CXCL8 neutralizing antibody (10 ng/mL). Scale bar = 200 μm. Data are shown as mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Human dermal lymphatic endothelial cells (hLECs) were purchased from ScienCell Research Laboratories and cultured under the manufacturer’s instructions.

Techniques: Control, Biomarker Discovery, RNA Sequencing, Quantitative RT-PCR, Expressing, Recombinant, Western Blot, Enzyme-linked Immunosorbent Assay, Migration

Journal: Oncotarget

Article Title: Podoplanin is a component of extracellular vesicles that reprograms cell-derived exosomal proteins and modulates lymphatic vessel formation

doi: 10.18632/oncotarget.7445

Figure Lengend Snippet: A. Quantitative evaluation of the length of tubes per field formed by HLECs seeded on Matrigel-coated wells untreated (Control) or treated with MDCK-CMV and MDCK-PDPN crude EXOs (40 μg/ml) for 2 h and 4 h. A representative experiment out of two is presented. B, C. Representative micrographs (B) and quantitative evaluation of the number of closed capillary-like structures per field (C) formed by HLECs seeded on Matrigel-coated wells untreated (Control) or treated with MDCK-CMV and MDCK-PDPN crude EXOs (40 μg/ml) for 6 h. EXOs were preincubated with mAb NZ1 (0.5 μg/ml and 1 μg/ml) recognizing the extracellular domain of PDPN or control IgG (1 μg/ml), as indicated, for 1h at 4°C. Bar, 100 μm. * p < 0.05. A representative experiment out of two is presented.

Article Snippet: In vitro formation of capillary-like structures was performed on growth factor-reduced Matrigel (Corning) with primary human umbilical vein endothelial cells (HUVECs) and human dermal lymphatic microvascular endothelial cells (HLECs) purchased from Lonza.

Techniques: Control

Journal: Signal Transduction and Targeted Therapy

Article Title: Anti-lymphangiogenesis for boosting drug accumulation in tumors

doi: 10.1038/s41392-024-01794-4

Figure Lengend Snippet: Anti-lymphangiogenesis activity of anlotinib and SAR131675 both in vitro and in vivo. a – c Anlotinib inhibited the proliferation ( a ), migration ( b ), and tubule network formation ( c ) of hLECs ( n = 4). Scale bar, 200 μm in ( b ) and 1000 μm in ( c ). d – f SAR131675 inhibited the proliferation ( d ), migration ( e ), and tubule network formation ( f ) of hLECs ( n = 4). Scale bar, 200 μm in ( e ) and 1000 μm in ( f ). g Quantification of the recovered area by hLECs migration as shown in ( b ) ( n = 3). h Quantification of the recovered area by hLECs migration as shown in and ( e ) ( n = 3). i Quantification of tube lengths as shown in ( c ) ( n = 3). j Quantification of tube lengths as shown in ( f ) ( n = 3). k Immunohistochemistry staining for LVs (LYVE-1, brown) in 4T1 tumor sections from mice treated with saline, anlotinib or SAR131675. Scale bar, 40 μm. l Quantification of tumor LV density ( n = 9; images were from three mice per group). m Typical images of inguinal LNs from different groups. Scale bar, 0.5 cm. The data are presented as the mean ± s.d. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: The human lymphatic endothelial cell line (hLECs) was purchased from iCell, China.

Techniques: Activity Assay, In Vitro, In Vivo, Migration, Immunohistochemistry, Staining, Saline