cells primary hlecs  (ScienCell)

Journal: Nature Communications

Article Title: APOA1 binding protein promotes lymphatic cell fate and lymphangiogenesis by relieving caveolae-mediated inhibition of VEGFR3 signaling

doi: 10.1038/s41467-025-60611-w

Figure Lengend Snippet: a TEM analysis of control and recombinant AIBP-treated hLECs. Arrows depict caveolae. b Quantification of caveolae in ( a ). n = 35 (control) and n = 36 (AIBP) cells. Data are Mean ± SE; unpaired two-sided t -test with Welch’s correction. c TEM analysis of caveolae in ECs of the cardinal vein from control and apoa1bp2 −/− zebrafish. Arrows indicate closed caveolae and arrowheads show open caveolae. d Quantification of caveolae in cardinal vein ECs. n = 27 (control) and n = 29 ( apo1bp2 −/− ) cells. Data are Mean ± SE; unpaired two-sided t -test with Welch’s correction. e Scheme illustration of 4 hydroxy-tamoxifen (4OHT)-induced ubiquitous mEos2-APOA1 expression. f Representative images of control and mEos2-APOA1 expressed embryos after 4OHT treatment. Embryos were imaged at 2 dpf. The white dashed line demarcates the control animals. The results are representative of 3 independent repeats. g Photoconversion and vascular circulation of mEos2-APOA1. The head regions of embryos were exposed to UV light for 1 min to induce photoconversion. h Analysis of mEos2-APOA1 secretion. The specified tail region was imaged, and the maximum RFP-to-GFP signal ratio within the ISV lumen was quantified. n = 8 intersegmental vessels from 2 embryos. Data are presented as mean ± SEM, analyzed using one-way ANOVA with Tukey’s post-hoc test. i Maxi-projection confocal images of Prox1 + cells in the apoa1bp −/− ; fli1a:egfp zebrafish with control (mEos2-APOA1) and mEos2-APOA1 overexpression (ubi:Gal4-ERT2+mEos2-APOA1) at 4 dpf and immunostained with GFP (green) and Prox1 (red) antibodies. Arrowheads show the Prox1 + LECs in TD. j Quantitative data of Prox1 + LEC in TD (7 somites). n = 10 ( mEos-APOA1 ) and n = 11 ( ubi:Ert2-Gal4; mEos-APOA1 ) embryos. Data are Mean ± SE; unpaired two-sided t -test with Welch’s correction. CV cardinal vein. Scale bar: 400 nm in a and 500 nm in c ; 100 µm in ( f , g , i ). Source data are prov i ded as a file.

Article Snippet: Primary human dermal lymphatic microvascular endothelial cells (hLECs) were purchased from PromoCell (Cat # C-12216 and C-12217).

Techniques: Control, Recombinant, Expressing, Over Expression

Journal: Nature Communications

Article Title: APOA1 binding protein promotes lymphatic cell fate and lymphangiogenesis by relieving caveolae-mediated inhibition of VEGFR3 signaling

doi: 10.1038/s41467-025-60611-w

Figure Lengend Snippet: a − d Effect of MβCD on VEGFR3 phosphorylation. a hLECs were growth factor-starved, and treated with 10 mM MβCD for 5, 15, and 30 min, and the resulting cells were further stimulated with 100 ng/mL VEGFC. The resulting cells were lysed and blotted using CAV-1, VEGFR3, GAPDH antibodies. b Quantitative analysis of panel a. Mean ± SD, n = 3 repeats; two-way ANOVA with Dunnett’s post-hoc test. c , hLECs were treated as in panel a. and cell lysates were immunoprecipitated using VEGFR3 antibody. Immunoblotting was performed using anti-phosphotyrosine (4G10) and VEGFR3 antibodies. d Quantitative analysis of ( c ). n = 3 repeats. Data are presented as mean ± SD and were analyzed using one-way ANOVA with Tukey’s post-hoc test. e , f Effect of AIBP treatment on VEGFR3 distribution in caveolar fractions. e hLECs were incubated with either recombinant AIBP or vehicle control in EBM2 supplemented with 10% FBS for 2 h, and the cells were subjected to sucrose gradient ultracentrifugation. n = 3 repeats. The resulting fractions were collected for Western blot analysis as indicated. Tx treatment; cav: caveolar fraction; n.c non-caveolar fraction. f Quantitative data of ( e ). Mean ± SD; two-way ANOVA with Sidak’s post-hoc test. n = 3 repeats. g , h Effect of AIBP and HDL co-treatment on VEGFR3 signaling. g hLECs were growth factor-starved and treated with HDL, AIBP, or HDL and AIBP in combination, and further stimulated with VEGFC. The resulting cells were lysed and immunoblotted as indicated. h Quantitative data of ERK and AKT activation. Mean ± SD; two-way ANOVA with Tukey’s post-hoc test. n = 3 repeats. Ctrl: control. i Maxi-projection confocal images of Prox1 + and pErk1/2 + cells in the apoa1bp −/− ; fli1a:egfp zebrafish at 36 hpf following immunostaining using GFP, pErk1/2, and Prox1 antibodies. Dorsal (DA) aorta and cardinal vein (CV) were imaged. Arrows show the Prox1 + LECs with pErk1/2 expression. j Quantitative data of pErk1/2 intensity in Prox1 + LECs. Data are Mean ± SE; unpaired two-sided t -test with Welch’s correction. n = 146 (control) and n = 166 ( apoa1bp −/− ) cells. Scale bar: 50 µm. Source data are provided as a file.

Article Snippet: Primary human dermal lymphatic microvascular endothelial cells (hLECs) were purchased from PromoCell (Cat # C-12216 and C-12217).

Techniques: Phospho-proteomics, Immunoprecipitation, Western Blot, Incubation, Recombinant, Control, Activation Assay, Immunostaining, Expressing

Journal: Nature Communications

Article Title: APOA1 binding protein promotes lymphatic cell fate and lymphangiogenesis by relieving caveolae-mediated inhibition of VEGFR3 signaling

doi: 10.1038/s41467-025-60611-w

Figure Lengend Snippet: a Conserved CAV-1 binding site on VEGFR3 in human (Hu), mouse (Ms), and zebrafish (Zf). The conserved amino acids are shown in blue. b Co-immunoprecipitation of endogenous VEGFR3 and CAV-1 in hLECs. Lysates from two 10 cm confluent plates of hLECs were combined, then equally divided for immunoprecipitation using VEGFR3 antibody or control protein A beads. The samples were subsequently immunoblotted for CAV-1 and VEGFR3. c , d VEGFR3 AAA loses its binding to CAV-1. c hLECs were transfected with control EGFP, VEGFR3-EGFP (R3), or VEGFR3 AAA -EGFP (R3 AAA ) using lentivirus-mediated gene transduction. After 72 hours, the resulting cells were lysed and immunoprecipitated with GFP antibody conjugated to agarose beads and immunoblotted using GFP and CAV-1 antibodies. d The input lysates were immunoblotted using GFP, CAV1, or GAPDH antibody as indicated. e Localization of VEGFR3 and VEGFR3 AAA in caveolae. hLECs were transduced with VEGFR3-APEX2 or VEGFR3 AAA -APEX2 Lenti-viral particles, and after 72 h, cells were fixed with 2.5% glutaraldehyde, stained using DAB substrate kit, and pelleted for TEM analysis. An enlarged view of a single caveola, highlighted with a white box, is shown in the top left corner of each image. f – h hLECs were transduced using lentivirus, and the resulting cells were growth factor starved and treated with 100 ng/mL VEGFC for 20 min, cells were then lysed and immunoblotted as indicated. R3/R3 AAA -EGFP denotes detection using GFP antibody. Quantitative data of VEGFR3 activation ( g ), ERK activation ( i ), and AKT activation ( j ) were shown. Mean ± SD; two-way ANOVA with Tukey’s post-hoc test. n = 3 independent repeats in g , i , j . Endg: endogenous. Scale bar: 400 nm. Source data are provided as a file.

Article Snippet: Primary human dermal lymphatic microvascular endothelial cells (hLECs) were purchased from PromoCell (Cat # C-12216 and C-12217).

Techniques: Binding Assay, Immunoprecipitation, Control, Transfection, Transduction, Staining, Activation Assay

Journal: bioRxiv

Article Title: Fibroblasts regulate lymphatic barrier functions in a tissue dependent manner

doi: 10.1101/2025.04.17.649442

Figure Lengend Snippet: (A) Schematic diagram of transwell secretome experiment. ( B) TEER measurements of hLEC monolayers after 48 hours of treatment with dermal fibroblast secretomes with [NHDF(G)] and without growth factors [NHDF(B)]. ( C) Representative immunofluorescence images of VE-cadherin, claudin-5, and ZO-1 on hLECs following treatment with NHDF secretomes. (D) Quantification of VE-cadherin, claudin-5, and ZO-1 expression levels using ImageJ (FIJI). (E) Quantification of lymphatic markers LYVE-1 and PROX1 expression in hLEC monolayers treated with NHDF secretomes. (F) FITC–Dextran (4 kDa) transport assay across hLECs after NHDF secretome treatment. Data are presented as mean ± SEM from n = 6 independent experiments. Statistical analysis was performed using an unpaired two-tailed t -test; p > 0.05 = not significant (ns), p ≤ 0.0001 = extremely significant (****). For the permeability (transport) assay ( n = 3, representative), statistical significance was determined by two-way ANOVA followed by appropriate post hoc test for multiple comparisons.

Article Snippet: Human primary dermal lymphatic endothelial cells (hLECs) (PromoCell, C-12217) were seeded onto the bottom side of Transwell inserts (1 μm pore size, Falcon®, 353103), coated with 50 μg/mL rat tail collagen I (Corning, 354236).

Techniques: Immunofluorescence, Expressing, Transport Assay, Two Tailed Test, Permeability

Journal: bioRxiv

Article Title: Fibroblasts regulate lymphatic barrier functions in a tissue dependent manner

doi: 10.1101/2025.04.17.649442

Figure Lengend Snippet: (A) Schematic diagram of transwell co-culture experiment. (B ) Schematic diagram of cells seedings and co-culture. (C) TEER measurements of hLEC monolayers after 48 hours of co-culture with dermal fibroblast with [NHDF(G)] and without growth factors [NHDF(B)]. (D) Representative immunofluorescence images of VE-cadherin, and ZO-1 on hLECs following co-culture with NHDFs. (E) Quantification of VE-cadherin and ZO-1 expression levels using ImageJ (FIJI). (F) FITC–Dextran (4 kDa) transport assay across hLECs after co-culture with NHDFs. Data are presented as mean ± SEM from n = 6 independent experiments. Statistical analysis was performed using an unpaired two-tailed t -test; p > 0.05 = not significant (ns), p ≤ 0.0001 = extremely significant (****). For the permeability (transport) assay ( n = 5, representative), statistical significance was determined by two-way ANOVA followed by appropriate post hoc test for multiple comparisons.

Article Snippet: Human primary dermal lymphatic endothelial cells (hLECs) (PromoCell, C-12217) were seeded onto the bottom side of Transwell inserts (1 μm pore size, Falcon®, 353103), coated with 50 μg/mL rat tail collagen I (Corning, 354236).

Techniques: Co-Culture Assay, Immunofluorescence, Expressing, Transport Assay, Two Tailed Test, Permeability

Journal: bioRxiv

Article Title: Fibroblasts regulate lymphatic barrier functions in a tissue dependent manner

doi: 10.1101/2025.04.17.649442

Figure Lengend Snippet: (A) Quantitative analysis of ZO-1 distribution and organization in hLEC monolayers using JAnaP after 48 hours of co-culture with NHDFs. (B) Immunoblot analysis of total ZO-1 (220 kDa) and VE-cadherin (120 kDa) protein expression levels in hLECs following co-culture. (C) RT-qPCR analysis of mRNA expression levels of ZO-1 and VE-cadherin in hLECs under the same conditions. Data are presented as mean ± SEM from n > 2 independent experiments. Statistical analysis was performed using an unpaired two-tailed t -test; p > 0.05 = not significant (ns), p ≤ 0.0001 = extremely significant (****).

Article Snippet: Human primary dermal lymphatic endothelial cells (hLECs) (PromoCell, C-12217) were seeded onto the bottom side of Transwell inserts (1 μm pore size, Falcon®, 353103), coated with 50 μg/mL rat tail collagen I (Corning, 354236).

Techniques: Co-Culture Assay, Western Blot, Expressing, Quantitative RT-PCR, Two Tailed Test

Journal: bioRxiv

Article Title: Fibroblasts regulate lymphatic barrier functions in a tissue dependent manner

doi: 10.1101/2025.04.17.649442

Figure Lengend Snippet: (A) TEER measurements of hLEC monolayers after 48 hours of treatment with NHLF secretomes or fibroblast media. (B) Representative immunofluorescence images of VE-cadherin, and ZO-1 on hLECs following treatment with NHLF secretome. (C) Quantification of VE-cadherin and ZO-1 expression levels using ImageJ (FIJI). (D) FITC–Dextran (4 kDa) transport assay across hLECs after treatment with 10, 25 and 50% NHLF secretome. Data are presented as mean ± SEM from n = 6 independent experiments. Statistical analysis was performed using an unpaired two-tailed t -test; p > 0.05 = not significant (ns), p ≤ 0.0001 = extremely significant (****). For the transport assay ( n = 3, representative), statistical significance was determined by two-way ANOVA followed by appropriate post hoc test for multiple comparisons.

Article Snippet: Human primary dermal lymphatic endothelial cells (hLECs) (PromoCell, C-12217) were seeded onto the bottom side of Transwell inserts (1 μm pore size, Falcon®, 353103), coated with 50 μg/mL rat tail collagen I (Corning, 354236).

Techniques: Immunofluorescence, Expressing, Transport Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Fibroblasts regulate lymphatic barrier functions in a tissue dependent manner

doi: 10.1101/2025.04.17.649442

Figure Lengend Snippet: (A) TEER measurements of hLEC monolayers after 4 hours of thrombin treatment followed by 48 hours of co-culture with dermal fibroblast (NHDF(G) ( n = 3 ). (B) Total cell counts ( n = 6 ). (C) FITC–Dextran (4 kDa) transport assay across hLECs after 4 hours of thrombin stimulation and/or NHDF co-culture for 48 hours. (D) Representative immunofluorescence images of ZO-1 and VE-cadherin on hLECs with and without thrombin treatment. Data are presented as mean ± SEM from n = 3-6 independent experiments. Statistical analysis was performed using an unpaired two-tailed t -test; p > 0.05 = not significant (ns), p ≤ 0.0001 = extremely significant (****). For the transport assay, statistical significance was determined by two-way ANOVA followed by appropriate post hoc test for multiple comparisons.

Article Snippet: Human primary dermal lymphatic endothelial cells (hLECs) (PromoCell, C-12217) were seeded onto the bottom side of Transwell inserts (1 μm pore size, Falcon®, 353103), coated with 50 μg/mL rat tail collagen I (Corning, 354236).

Techniques: Co-Culture Assay, Transport Assay, Immunofluorescence, Two Tailed Test

Journal: JCI Insight

Article Title: Mycobacterium tuberculosis cords within lymphatic endothelial cells to evade host immunity

doi: 10.1172/jci.insight.136937

Figure Lengend Snippet: (A) Images of primary hLECs infected with GFP expressing M. tuberculosis for 2 to 72 hours. Over time, M. tuberculosis grows and forms large intracellular cords. Nuclei are stained with DAPI (blue) and F-actin is stained by rhodamine phalloidin (red). (B) 3D reconstruction of Z-stacks taken of an intracellular cord from (A). Various angles are shown to confirm that the cord is completely encapsulated within the host cell. (C) Measurement of the intracellular cords over time in hLECs (using the Feret diameter; see Supplemental Figure 1) showing that the cords elongated up to a maximum of 150 μm. The numbers of bacterial clusters analyzed were 418 (2 hours), 233 (24 hours), 814 (48 hours), and 618 (72 hours), obtained from 3 independent experiments. One-way ANOVA with Tukey’s multiple comparisons tests: ***P < 0.001. (D) Image of A549 cells infected with M. tuberculosis-EGFP for 72 hours showing an intracellular cord looping around the nucleus. Nuclei are stained with DAPI (blue) and F-actin is stained with rhodamine phalloidin (red). (E) Intracellular cord formation after 72 hours was also observed in hLECs infected with representative strains from 3 other M. tuberculosis lineages: N0072 (lineage 1), N0145 (lineage 2), and N0024 (lineage 3). Images displayed in D and E are representative of at least 3 independent experiments. (F) Tissue section of a granuloma present in a human lymph stained for AFB. Zoomed region shows association of M. tuberculosis cords with cells (black boxes). Representative histological sections from human patients after lymph node tissue resection surgery were stained for PDPN, M. tuberculosis, and nuclei (DAPI). Scale bar: 1 mm. White boxes delimit the zoomed regions displayed on the right-hand side. Arrows indicate the presence of M. tuberculosis cords within PDPN+ cells. Scale bar: 20 μm. hLECs, human lymphatic endothelial cells; AFB, acid fast bacilli; PDPN, podoplanin.

Article Snippet: Cells Primary hLECs taken from inguinal lymph nodes (ScienCell Research Laboratories, catalog 2500) were cultured according to the manufacturer’s instructions up to passage 5 as described fully in ref. 19 .

Techniques: Infection, Expressing, Staining

Journal: Cell reports

Article Title: Complementary Wnt Sources Regulate Lymphatic Vascular Development via PROX1-Dependent Wnt/β-Catenin Signaling

doi: 10.1016/j.celrep.2018.09.049

Figure Lengend Snippet: (A) HLECs were exposed to oscillatory shear stress (OSS) for the indicated number of hours. Subsequently, the cells were harvested and analyzed by western blotting using the indicated antibodies. Phosphorylation of LRP6 is increased by OSS. (B and C) HLECs were cultured under OSS with recombinant DKK1, which inhibits the interaction between Wnt ligands and LRP co-receptors (B), or LGK-974, which inhibits the secretion of Wnt ligands from cells (C). Subsequently, RNA was extracted, and the expression of AXIN2 , FOXC2 , and GATA2 was evaluated by real-time qPCR analysis. The data were normalized to GAPDH . Both DKK1 and LGK-974 inhibit OSS-induced expression of AXIN2, FOXC2 , and GATA2 . (D and E) E15.5 LVVs are observed in both Lyve1Cre ; Wls f/f (E) and control (D) littermate embryos (arrows). (F–I) Whole-mount immunohistochemistry on the dorsal skin of E16.5 wild-type (F) and Lyve1Cre;Wls f/f (G) embryos reveals normal lymphatic vessels. The diameters of lymphatic vessels (H) and their migration toward the dorsal midline (I) are not different between E17.5 control and mutant embryos. (J–L)Normal-looking LVs are observed inmesenteric lymphatic vessels of P2 control (J, arrow) and Lyve1Cre;Wls f/f mice (K,arrow). The number of E18.5 LVs is not changed in Lyve1-Cre;Wls f/f mice (L). LVV, lymphovenous valve; LV, lymphatic valve;IJV, internal jugular vein; SCV, subclavian vein. Scale bars, 100 mm (D–G) and 200 mm (J and K). Statistics: (A)–(C), n = 3; (D)–(L), n = 4 per genotype per stage; **p < 0.01, ***p < 0.001, ****p < 0.0001. Error bars in graphs represent ± SEM.

Article Snippet: We obtained primary human lymphatic endothelial cells (HLECs) from Lonza and primary human umbilical vein ECs (HUVECs) from Thermo Fisher Scientific.

Techniques: Shear, Western Blot, Phospho-proteomics, Cell Culture, Recombinant, Expressing, Control, Immunohistochemistry, Migration, Mutagenesis

Journal: Cell reports

Article Title: Complementary Wnt Sources Regulate Lymphatic Vascular Development via PROX1-Dependent Wnt/β-Catenin Signaling

doi: 10.1016/j.celrep.2018.09.049

Figure Lengend Snippet: (A) HLECs were cultured under OSS with or without the Wnt agonist BIO. Subsequently, cells were lysed, and western blot was performed for the indicated molecules. Both OSS and BIO enhance FOXC2 expression. BIO enhances FOXC2 expression to a greater extent compared with OSS. Additionally, a much stronger expression of FOXC2 is observed in HLECs cultured with both OSS and BIO. (B–I) W/s was simultaneously deleted from LECs and vascular smooth muscle cells by Lyve1-Cre and SM22-Cre. The control and mutant embryos were harvested at the indicated developmental time points and analyzed by immunohistochemistry on frontal cryosections (B and C) or by whole-mount immunohistochemistry(D–I). (B and C) LVVs are observed at the junction of lymph sacs (LSs), IJV, and subclavian vein (SCV) in control (B, arrows) but not in SM22-Cre; Lyve1-Cre;W/s f/f embryos (C). (D–G) LVs are observed in the mesenteric lymphatic vessels of control embryos (D, arrows, and F). LVs are mostly absent from the mesenteric lymphatic vessels of SM22-Cre; Lyve1-Cre;W/s f/f embryos (E and G). (H–I´) (H) and (I) are higher-magnification images of the boxed areas in (H´) and (I´), respectively. The lymphatic vessels of the dorsal skin are dilated in mutant embryos. The distance between the opposing migrating fronts is higher in SM22-Cre; Lyve1-Cre;W/s f/f embryos (magenta bars in I’). Additionally, fewer branchpoints are observed in the lymphatic vessels of mutant embryos. (J–M) The number of mesenteric LVs per lymphatic vessel is quantified in (J). The lymphatic vessels of the dorsal skin were analyzed, and the distance between the migrating fronts (K), diameters of lymphatic vessels (L), and the number of branchpoints (M) were quantified. Scale bars, 100 mM (B, C, and F–I), 200 mM (H’ and I’), and 250 mM (D and E). Statistics: (A), n = 3; (B)–(M), n = 4 per genotype; ***p < 0.001, ****p < 0.0001. Error bars in graphs represent ± SEM.

Article Snippet: We obtained primary human lymphatic endothelial cells (HLECs) from Lonza and primary human umbilical vein ECs (HUVECs) from Thermo Fisher Scientific.

Techniques: Cell Culture, Western Blot, Expressing, Control, Mutagenesis, Immunohistochemistry

Journal: Cell reports

Article Title: Complementary Wnt Sources Regulate Lymphatic Vascular Development via PROX1-Dependent Wnt/β-Catenin Signaling

doi: 10.1016/j.celrep.2018.09.049

Figure Lengend Snippet: (A and B) OSS and Wnt/β-catenin signaling do not enhance FOXC2 and GATA2 expression in human umbilical vein endothelial cells (HUVECs). Primary human lymphatic endothelial cells (HLECs) or HUVECs were cultured under OSS for 48 hr (A) or in the presence of recombinant Wnt3a (rWnt3a) for 24 hr (B). Subsequently, RNA was extracted and analyzed by real-time qPCR for AXIN2, FOXC2 , and GATA2 . The expression levels were normalized to that of GAPDH. (C and D) PROX1 provides competence to HUVECs to respond to OSS and Wnt/β-catenin signaling and enhance the expression of FOXC2 and GATA2. HUVECs were infected with lentiviral particles expressing GFP or human PROX1 for 48 hr. Subsequently, the cells were exposed to (C) OSS for 48 hr or to (D) the Wnt agonist BIO for 12 hr. (C) PROX1-expressing HUVECs were cultured under static or OSS conditions with or without the Wnt antagonist iCRT3. Western blot was performed to quantify FOXC2 expression. (D) PROX1-expressing HUVECs were cultured with BIO or DMSO for 24 hr, and the expression of FOXC2 and GATA2 was quantified by real-time qPCR. (E and F) PROX1 is necessary for OSS- and Wnt/β-catenin signaling-mediated expression of FOXC2 and GATA2 in HLECs. HLECs were infected with lentiviral particles expressing shRNAs that target GFP (sh-Con) or PROX1 (sh-P1#1 and sh-P1#2) for 48 hr. Subsequently, HLECs were additionally cultured under OSS for 48 hr (E) or with rWnt3a for 24 hr (F). Real-time qPCR was performed to quantify the expression of FOXC2 and GATA2 . Statistics: n = 3 for all experiments. **p < 0.01, ***p < 0.001, ****p < 0.0001. Error bars in graphs represent ± SEM.

Article Snippet: We obtained primary human lymphatic endothelial cells (HLECs) from Lonza and primary human umbilical vein ECs (HUVECs) from Thermo Fisher Scientific.

Techniques: Expressing, Cell Culture, Recombinant, Infection, Western Blot

Journal: Cell reports

Article Title: Complementary Wnt Sources Regulate Lymphatic Vascular Development via PROX1-Dependent Wnt/β-Catenin Signaling

doi: 10.1016/j.celrep.2018.09.049

Figure Lengend Snippet: (A) PROX1 enhances the expression of Wnt/β-catenin signaling target genes in HLECs. HLECs were infected with lentiviral particles expressing shRNAs that target GFP (sh-GFP) or PROX1 (shPROX1#1 and sh-PROX1#2) for 72 hr. Subsequently, RNA was extracted, and real-time qPCR was performed for the expression of Wnt/β-catenin target genes. (B–D) PROX1 synergizes with β-catenin to enhance Wnt/β-catenin signaling. (B) 293T cells were co-transfected with TOPFlashor FOPFlash luciferase reporters with PROX1- and or β-catenin-expressing vectors. The TCF/LEF binding sites of TOPFLASH are inactivated to generate FOPFlash as a negative control for Wnt/β-catenin signaling. (C and D) 293T cells were co-transfected with TOPFlash- and PROX1-expressing vectors together with AXIN-expressing (C) or ΔN-TCF7L2expressing (D) plasmids. DN-TCF7L2 cannot interact with β-catenin and functions as a dominant-negative mutant. A Renilla luciferase-expressing plasmid was used as an internal control, and luciferase activities were measured 36 hr after transfection. (E–G) PROX1 associates with β-catenin. (E) 293T cells were transfected with Myc-tagged β-catenin and FLAG-tagged PROX1 plasmids. 48 hr later, the cell lysate was subjected to a co-immunoprecipitation assay using anti-FLAG antibody or anti-Myc antibody. The precipitates were probed by western blot using the indicated antibodies. (F) HLEC lysate was immunoprecipitated using anti-PROX1 antibody and probed by western blot using anti-PROX1 or anti-β-catenin antibodies. (G) HLECs were treated with BIO for the indicated number of hours. The lysates were analyzed as in (F). (H) HLECs were treated with DMSO or BIO (0.5 μM) for 3 hr, and ChIP was performed using anti-PROX1 antibody. qPCR was performed using primers flanking the TCF/LEF-binding site in the 3.5 kb location. As a negative control, primers flanking a TCF/LEF-binding site that is located at a more upstream location (5.5 kb) were used. Statistics: n = 3 for all experiments. **p < 0.01, ***p < 0.001. Error bars in graphs represent ± SEM.

Article Snippet: We obtained primary human lymphatic endothelial cells (HLECs) from Lonza and primary human umbilical vein ECs (HUVECs) from Thermo Fisher Scientific.

Techniques: Expressing, Infection, Transfection, Luciferase, Binding Assay, Negative Control, Dominant Negative Mutation, Plasmid Preparation, Control, Co-Immunoprecipitation Assay, Western Blot, Immunoprecipitation

Journal: Cell reports

Article Title: Complementary Wnt Sources Regulate Lymphatic Vascular Development via PROX1-Dependent Wnt/β-Catenin Signaling

doi: 10.1016/j.celrep.2018.09.049

Figure Lengend Snippet: (A and B) E17.5 embryos were frontally sectioned and analyzed using the indicated antibodies. Lymphovenous valves (arrows) and venous valves (yellow arrowheads) are present at the junction of the IJV, external jugular vein (EJV), SCV, and LSs of control (A) but not Tcf7l1 DN/DN (B) embryos. (C–K) The dorsal skin of E17.5 embryos was analyzed by whole-mount immunohistochemistry. (C) The distance between the lymphatic vessel migrating fronts is significantly increased in Tcf7l1 ΔN/ΔN embryos. A representative image is shown in . (D–K) Compared to controls (D, G, and J), the lymphatic vessels of Tcf7l1 ΔN/ΔN are dilated (E and F) and have abnormal αSMA + vascular SMC coverage (H, yellow arrowheads, and I). PROX1 high ; FOXC2 high LVs are observed in wild-type (G and J, arrows) but not in Tcf7l1 ΔN/ΔN embryos (K). LVs are rarely observed in the mesenteric lymphatic vessels of E17.5 Tcf7l1 ΔN/ΔN embryos. Representative images are shown in . (M) HLECs were infected with lentiviral particles expressing GFP or human ΔN-TCF7L1 for 24 hr. ΔN-TCF7L1 cannot interact with α-catenin and functions as a dominant-negative mutant. Subsequently, the cells were exposed to OSS for 48 hr, and the expression of Axin2, FOXC2, and GATA2 was measured by real-time qPCR. (N) 293T cells were transfected with PROX1 together with FLAG-tagged TCF7L1 or ΔNTCF7L1 plasmids. After 48 hr, an immunoprecipitation assay was performed using anti-PROX1 antibody. The precipitate was probed by western blot using the indicated antibodies to determine the interaction between PROX1, TCF7L1, and endogenous β-catenin. PROX1 interacts with TCF7L1 but not ΔN- TCF7L1. Furthermore, TCF7L1, but not ΔN-TCF7L1, enhances the interaction between PROX1 and β-catenin. Scale bars, 200 μM (A, B, G, and H), 100 μM (J and K), and 50 μmM (D and E). Statistics: (A)–(L), n = 4 per genotype; (M) and (N), n = 3; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Error bars in graphs represent ± SEM.

Article Snippet: We obtained primary human lymphatic endothelial cells (HLECs) from Lonza and primary human umbilical vein ECs (HUVECs) from Thermo Fisher Scientific.

Techniques: Control, Immunohistochemistry, Infection, Expressing, Dominant Negative Mutation, Transfection, Immunoprecipitation, Western Blot

Journal: Cell reports

Article Title: Complementary Wnt Sources Regulate Lymphatic Vascular Development via PROX1-Dependent Wnt/β-Catenin Signaling

doi: 10.1016/j.celrep.2018.09.049

Figure Lengend Snippet:

Article Snippet: We obtained primary human lymphatic endothelial cells (HLECs) from Lonza and primary human umbilical vein ECs (HUVECs) from Thermo Fisher Scientific.

Techniques: Virus, Recombinant, Electron Microscopy, Transfection, SYBR Green Assay, cDNA Synthesis, Bicinchoninic Acid Protein Assay, Software

Journal: Drug Design, Development and Therapy

Article Title: Inhibition of lymphangiogenesis in vitro and in vivo by the multikinase inhibitor nintedanib

doi: 10.2147/dddt.s130297

Figure Lengend Snippet: Figure 1 Effect of BIBF 1120 (nintedanib) on growth factor-induced proliferation of HLECs. Notes: (A) VEGF-C (50 ng/mL) enhanced proliferation of HLECs significantly. The enhanced effect was significantly reduced by 0.01 μM nintedanib (P,0.05) and almost eliminated by 0.1 μM nintedanib (P,0.01). (B) PDGF-BB (50 ng/mL) enhanced proliferation of HLECs significantly. The enhanced effect was significantly reduced by 0.01 μM nintedanib (P,0.05) and almost eliminated by 0.1 μM nintedanib (P,0.01). (C) A concentration of 50 ng/mL of bFGF enhanced proliferation of HLECs significantly. The enhanced effect was significantly reduced by 0.1 μM nintedanib (P,0.05) and almost eliminated by 1 μM nintedanib (P,0.01). *P-value ,0.05. **P-value ,0.01. Abbreviations: HLECs, human lymphatic endothelial cells; VEGF-C, vascular endothelial growth factor-C; PDGF-BB, platelet-derived growth factor-BB; bFGF, basic fibroblast growth factor; OD, optical density.

Article Snippet: Animal experiments were performed in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research; the study was approved by Fudan University’s Animal Ethical Committee. cell culture Primary human lymphatic endothelial cells (HLECs) were purchased from PromoCell (Heidelberg, Germany) and cultured in endothelial basal medium (EBM; Lonza, Walkersville, MD, USA) supplemented with 10% fetal calf serum (FCS; Biowest, Riverside, MO, USA) and other supplements, as previously described.24 HLECs at passages 4–8 were used in this study. nintedanib Nintedanib was purchased from Selleckchem (Houston, TX, USA).

Techniques: Concentration Assay, Derivative Assay

Journal: Drug Design, Development and Therapy

Article Title: Inhibition of lymphangiogenesis in vitro and in vivo by the multikinase inhibitor nintedanib

doi: 10.2147/dddt.s130297

Figure Lengend Snippet: Figure 2 BIBF 1120 (nintedanib) suppressed growth factor-induced migration of HLECs. Notes: (A) Cells treated with 0.1% DMSO were used as controls. (B1 and B2) VEGF-C (50 ng/mL) promoted migration of HLECs significantly, and the enhanced effect was significantly reduced by 0.1 μM nintedanib. (C1 and C2) PDGF-BB (50 ng/mL) promoted migration of HLECs significantly, and the enhanced effect was significantly reduced by 0.1 μM nintedanib. (D1 and D2) A concentration of 50 ng/mL of bFGF enhanced migration of HLECs significantly, and the enhanced effect was significantly reduced by 0.1 μM nintedanib. (E) The number of migrated cells stimulated by VEGF-C, PDGF-BB, or bFGF at a concentration of 50 ng/mL in the presence or absence of 0.1 μM nintedanib. A–D at 200× magnification; scale bar =50 µm. Abbreviations: HLECs, human lymphatic endothelial cells; DMSO, dimethyl sulfoxide; VEGF-C, vascular endothelial growth factor-C; PDGF-BB, platelet-derived growth factor-BB; bFGF, basic fibroblast growth factor.

Article Snippet: Animal experiments were performed in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research; the study was approved by Fudan University’s Animal Ethical Committee. cell culture Primary human lymphatic endothelial cells (HLECs) were purchased from PromoCell (Heidelberg, Germany) and cultured in endothelial basal medium (EBM; Lonza, Walkersville, MD, USA) supplemented with 10% fetal calf serum (FCS; Biowest, Riverside, MO, USA) and other supplements, as previously described.24 HLECs at passages 4–8 were used in this study. nintedanib Nintedanib was purchased from Selleckchem (Houston, TX, USA).

Techniques: Migration, Concentration Assay, Derivative Assay

Journal: Drug Design, Development and Therapy

Article Title: Inhibition of lymphangiogenesis in vitro and in vivo by the multikinase inhibitor nintedanib

doi: 10.2147/dddt.s130297

Figure Lengend Snippet: Figure 3 BIBF 1120 (nintedanib) inhibited growth factor-induced tube formation of HLECs. Notes: (A) Cells treated with 0.1% DMSO were used as controls. (B1 and B2) VEGF-C (50 ng/mL) enhanced tube formation of HLECs significantly, and the enhanced effect was significantly reduced by 0.1 μM nintedanib. (C1 and C2) PDGF-BB (50 ng/mL) enhanced tube formation of HLECs significantly, and the enhanced effect was significantly reduced by 0.1 μM nintedanib. (D1 and D2) A concentration of 50 ng/mL of bFGF enhanced tube formation of HLECs significantly, and the enhanced effect was significantly reduced by 0.1 μM nintedanib. (E) The area of the tube structure after being stimulated by VEGF-C, PDGF-BB, and bFGF at a concentration of 50 ng/mL in the presence or absence of 0.1 μM nintedanib. (F) The length of the tube structure after being stimulated by VEGF-C, PDGF-BB, and bFGF at a concentration of 50 ng/mL in the presence or absence of 0.1 μM nintedanib. A–D at 100× magnification. Abbreviations: HLECs, human lymphatic endothelial cells; DMSO, dimethyl sulfoxide; VEGF-C, vascular endothelial growth factor-C; PDGF-BB, platelet-derived growth factor-BB; bFGF, basic fibroblast growth factor.

Article Snippet: Animal experiments were performed in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research; the study was approved by Fudan University’s Animal Ethical Committee. cell culture Primary human lymphatic endothelial cells (HLECs) were purchased from PromoCell (Heidelberg, Germany) and cultured in endothelial basal medium (EBM; Lonza, Walkersville, MD, USA) supplemented with 10% fetal calf serum (FCS; Biowest, Riverside, MO, USA) and other supplements, as previously described.24 HLECs at passages 4–8 were used in this study. nintedanib Nintedanib was purchased from Selleckchem (Houston, TX, USA).

Techniques: Concentration Assay, Derivative Assay