cell lines human umbilical vein endothelial cells huvecs atcc pcs 100 010 human dermal microvascular endothelial cells  (ATCC)

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: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Activation of WNK1 signaling through Piezo1

doi: 10.1073/pnas.2513155122

Figure Lengend Snippet: Hypoosmotic stress activates OSR1 and SPAK through Piezo1. ( A ) Schematic of a cell responding to osmotic stress, either hyperosmotic or hypoosmotic, resulting in changes in membrane tension and Ca 2+ influx. ( B ) Immunoblot analysis of HDMECs treated with osmotic stress in the presence of DMSO or WNK463 (1 μM) for 5 min. The bar graph is presented as mean ± SD from three independent experiments. ** P < 0.01, *** P < 0.001 (unpaired two-tailed t test, nontreated vs. WNK463-treated); # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001 (one-way ANOVA with Tukey’s multiple comparison test within the DMSO group). ( C ) siCTRL- and siPiezo1-treated HDMECs were exposed to osmotic stress for 5 min and analyzed by immunoblotting using the indicated antibodies. The results are expressed as mean ± SD. ns, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001 (n = 3), unpaired two-tailed t test (siCTRL vs. siPiezo1). ( D and E ) Intracellular Ca 2+ levels were measured in Fluo-8 AM (2 µM)-labeled HDMECs exposed to isotonic, hypertonic (500 mM NaCl), or hypotonic [3:2 (v/v) deionized H 2 O/culture medium mixture] solutions, as indicated by the arrows. Data are expressed as mean ± SD (8 replicates, 2 independent experiments) ( D ). The graph represents the relative Fluo-8 AM intensity measured 3 min after osmotic stress exposure ( E ). Data are shown as mean ± SD. *** P < 0.001 (unpaired two-tailed t test, 8 replicates). ( F ) Ca 2+ -starved HDMECs, maintained in Ca 2+ /Mg 2+ -free HBSS (−Ca 2+ ) containing 0.1% FBS for 1 h, were exposed to osmotic stress in the Ca 2+ -free HBSS for 5 min. Cell lysates were analyzed by SDS-PAGE and immunoblotting. Data are shown as mean ± SD. ** P < 0.01; ns, not significant (n = 3). ( G ) Immunoblot analysis of HDMECs preincubated with DMSO or BAPTA-AM (10 μM) for 10 min, followed by exposure to osmotic stress for 5 min. The results are presented as mean ± SD. ns, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001 (n = 3), unpaired two-tailed t test (DMSO-treated vs. BAPTA-AM-treated).

Article Snippet: Primary human dermal microvascular endothelial cells (HDMECs) (CRL-3243, ATCC, Manassas, VA) were grown in complete MCDB 131 medium (15-100-CV, Corning, Corning, NY) supplemented with 10% FBS (F0926, MilliporeSigma, St Louis, MO), 1% L-glutamine (25-005-CI, Corning), 1% penicillin/streptomycin (SV30010, Thermo Fisher Scientific, Waltham, MA), 1 μg/mL hydrocortisone (H0888, MilliporeSigma), and 10 ng/mL epidermal growth factor (PHG0311, Thermo Fisher Scientific).

Techniques: Membrane, Western Blot, Two Tailed Test, Comparison, Labeling, SDS Page