Journal: The EMBO Journal

Article Title: Distinct roles of VE ‐cadherin for development and maintenance of specific lymph vessel beds

doi: 10.15252/embj.201798271

Figure Lengend Snippet: RNA isolation and quantitative real‐time PCR (qPCR) using TaqMan Assays

Article Snippet: Gene symbol Reference Angpt1 Mm01129232_m1 Angpt2 Mm00545822_m1 Cdh2 Mm01162497_m1 Cldn5 Mm00727012_s1 Ctgf Mm01192933_g1 Cyr61 Mm00487498_m1 Esam Mm00518378_m1 F11r (Jam1) Mm00554113_m1 Jam2 Mm00470197_m1 Jam3 Mm00499214_m1 Ocln Mm00500912_m1 Pecam1 Mm01242584_m1 Tek Mm00443243_m1 Tjp1 (Zo‐1) Mm00493699_m1 Trp73 Mm00660220_m1 Vegf‐C Mm01202430_m1 Vegf‐D Mm01131929_m1 Wwtr1 (Taz) Mm01289583_m1 Yap1 Mm01143263_m1 Open in a separate window RNA isolation and quantitative real‐time PCR (qPCR) using TaqMan Assays

Techniques: Isolation, Real-time Polymerase Chain Reaction

Journal: Nature Neuroscience

Article Title: Piezo1 regulates meningeal lymphatic vessel drainage and alleviates excessive CSF accumulation

doi: 10.1038/s41593-024-01604-8

Figure Lengend Snippet: a , Increased phosphorylation of CDH5 (Try 658), VEGFR2 (Tyr 1054/Tyr 1059) and VEGFR3 (Tyr 1230/Tyr 1231) in cultured primary human LECs in vitro treated with Yoda1 (0.5 µM, final). b , CDH5 immunofluorescence stains showing increased junctional gaps (arrowheads) in 2D LEC in vitro cultures after Yoda1 treatment at 0.5, 1 or 2 µM for 8 h; scale bars, 20 µm. c , Piezo1-dependent increase in drainage efficiency of engineered lymphatics in vitro. Primary LECs were transfected with control (scrambled) or Piezo1 short interfering RNA (siRNA) for 24 h, used to build lymphatics in 3D PDMS chips and treated with vehicle or Yoda1 (1 µM, final) to evaluate drainage capability. Detailed images for the engineered lymphatics are shown in Extended Data Fig. ; siCTR and vehicle, n = 7 independent experiments; siCTR and Yoda1, n = 8 independent experiments; siPiezo1 and vehicle, n = 8 independent experiments; siPiezo1 and Yoda1, n = 5 independent experiments. Data were analyzed by one-way ANOVA ( P = 0.0097) followed by a Bonferroni multiple comparison test. d , Lymphatic vessel contractility test. Surgically excised axillary collecting lymphatics were treated with vehicle, Yoda1 or l -NAME + Yoda1 at the indicated intraluminal pressures, and the percent vessel tone was measured. Additional functionality values are shown in Extended Data Fig. ; n = 6 independent samples. Data were analyzed by two-way repeated measures ANOVA ( P = 0.003) between treatments followed by Tukey’s multiple comparison test; P < 0.0001 between control and Yoda1; P = 0.0004 between control and l -NAME + Yoda1; P < 0.0001 between Yoda1 and l -NAME + Yoda1. e , Inhibition of Piezo1 downstream effectors suppresses Yoda1-induced promotion of brain tracer drainage. Fluorescent tracer (OVA-Green) was premixed with vehicle, Yoda1 (50 µM, final) or Yoda1 (50 µM, final) + inhibitors, such as l -NAME (eNOS), capivasertib (Cap; AKT), axitinib (Axi; VEGFR1–VEGFR3), SAR131675 (SAR; VEGFR3) or cabozantinib malate (Cab; VEGFR2; 258 µg ml –1 final concentration of each inhibitor), and i.c.m. injected into Prox1 -tdTomato mice (15 weeks old). After 45 min, LNs were collected and imaged; scale bars, 500 µm. f , g , Quantification of relative tracer intensity in cervical ( f ) and mandibular ( g ) LNs ( n = 7 mice per group). One data point represents the sum of the fluorescence intensity of LNs of both sides of a single mouse. Data were analyzed by one-way ANOVA ( P < 0.0001) followed by Tukey’s multiple comparison test. h , i , Protein expression levels of VEGF-C ( h ) and VEGF-D ( i ) were measured in the whole brains of mice that were treated with vehicle or Yoda1 (213 µg per kg (body weight) two times at 18 h and 90 min before tissue collection) or in the whole brains of control or Piezo1 TG_LEC mice that were i.p. injected with tamoxifen (50 mg per kg (body weight) twice, 3 days apart) at the age of 8 weeks. Pz1 TG , Piezo1 TG_LEC mice. Each data point represents one mouse ( n = 5 mice per group). Data were analyzed by two-tailed t -test and are presented as mean values ± s.e.m.

Article Snippet: Concentrations of VEGF-C and VEGF-D in the brain extracts were quantified using a mouse VEGF-C ELISA kit and a mouse VEGF-D ELISA kit (CUSABIO, CSB-E07361m and CSB-E07357m, respectively).

Techniques: Cell Culture, In Vitro, Immunofluorescence, Transfection, Small Interfering RNA, Comparison, Inhibition, Concentration Assay, Injection, Fluorescence, Expressing, Two Tailed Test

Journal: Open Medicine

Article Title: Transcription factor A, mitochondrial promotes lymph node metastasis and lymphangiogenesis in epithelial ovarian carcinoma

doi: 10.1515/med-2024-1089

Figure Lengend Snippet: Primer sequences for RT-qPCR

Article Snippet: Then, VEGF-A , VEGF-C , and VEGF-D were detected by ELISA kits ( VEGF-A kit, Multisciences, Hangzhou, China; VEGF-C kit and VEGF-D kit, CUSABIO, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Sequencing

Journal: Nature

Article Title: VEGF-C-driven lymphatic drainage enables brain tumor immunosurveillance

doi: 10.1038/s41586-019-1912-x

Figure Lengend Snippet: a-c RNAseq data of tumor tissue and healthy brain tissue from different regions of the tissue (TCGA (phs000178.v10.p8) and GTEX respectively (v6). a Expression profile of VEGF-A. b Expression profile of VEGF-C (GBM, n = 147; cortex, n = 133; amygdala, n = 81; Brodmann area 24&9, n = 215; C1 segment, n = 75; caudate nucleus, n = 135; cerebellar hemisphere, n = 115; cerebellum, n = 146; hippocampus, n = 103; hypothalamus, n = 101; nucleus accumbens, n = 125; putamen, n = 103; substantia nigra, n = 72; tibial nerve, n = 329). c RNAseq data of mice brain and GL261 tumors from mice brains were analyzed (n = 3 biologically independent samples). d ONCLNC ( Onclnc.org ) data of GBM patients stratified into two groups (VEGF-C low, lower 33%; VEGF-C hi, upper 33%; n = 50). e Kaplan Meier Survival curve of patients from e (n = 50). Data are mean ± S.D. *P < 0.05; **P < 0.01; ***P <0.001; ****P<0.0001 (two-tailed unpaired Student’s t-test, two-sided Log-rank Mantel-Cox test, Pearson’s correlation)

Article Snippet: Recombinant proteins (VEGF-A, VEGF-B, VEGF-C Cys156Ser, VEGF-D) were purchased from R&D systems.

Techniques: Expressing, Two Tailed Test