Journal: bioRxiv

Article Title: VEGF-A/C co-stimulation, without shear stress, triggers the polarization of lymphatic microvessels

doi: 10.1101/2025.08.13.670220

Figure Lengend Snippet: (A) Scheme of the silicone LµV shown in grey, and the fabrication protocol to produce a hollow tube in collagen gels represented in pink. After LEC seeding, the endothelial tissue is cultured for three days. (B) Optical micrograph of the LµV just after its fabrication and after two days of culture with or without stimulation with growth factors. (C) The LµV contour shown in red is segmented on each side of the vessel. (D) The graph shows the relative change of the contour length after 3 days of culture with or without growth factor stimulation. (E) Live imaging of a LµV shows the dynamic formation of sprouts in response to VEGF-A/C co-stimulation. Segmented sprouts are shown in red. (F) Maximum intensity projection (MIP) of confocal micrographs showing a sprout after 3 days of VEGF-A/C co-stimulation. Labels: phalloidin in gray and nucleus in cyan. Scale bars indicate 100 µm, except for the MIP, where it represents 50 µm.

Article Snippet: Human dermal lymphatic endothelial cells (HDLECLot 483Z001.2, PromoCell) were cultured in Microvascular Endothelial Cell Growth Medium-2 BulletKit (EGM-2, Lonza) in standard tissue culture incubators at 37°C, 95% humidity, and 5% CO2.

Techniques: Cell Culture, Imaging

Journal: Journal of Virology

Article Title: Kaposi's Sarcoma-Associated Herpesvirus Nonstructural Membrane Protein pK15 Recruits the Class II Phosphatidylinositol 3-Kinase PI3K-C2α To Activate Productive Viral Replication

doi: 10.1128/JVI.00544-18

Figure Lengend Snippet: Depletion of PI3K-C2α from infected endothelial cells leads to reduced KSHV lytic replication. HuARLT2-rKSHV cells were microporated with either a control siRNA (Scr), four individual siRNAs or a pool of these siRNAs against PI3K-C2α, or a single siRNA against K15, and the KSHV lytic cycle was induced 24 h later using a cocktail of RTA and SB. (A to C) Forty-eight hours after lytic induction, images for GFP and RFP expression were acquired (A), and the number of RFP-positive cells from five or more fields was quantified; cells were then lysed, and the expression level of the indicated viral proteins was assessed by Western blotting (C). Results are representative of data from two independent experiments. The scatter plot (B) represents the means ± standard deviations (SD) for five or more fields. Ordinary one-way analysis of variance (ANOVA) was used to determine P values. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (D and E) Primary lymphatic endothelial cells infected with KSHV for 2 weeks (LEC-rKSHV) were transfected with either the pooled PI3K-C2α siRNA or a scrambled control. After 72 h, cells and the culture supernatant were collected. The expression levels of the indicated proteins were analyzed by Western blotting (D), and the amount of released infectious virus was assayed by titrating the culture supernatant on HEK-293 cells (E). A Mann-Whitney test was used to determine P values. **, P < 0.01.

Article Snippet: Primary juvenile foreskin lymphatic endothelial cells (LECs; Lonza) were provided by Päivi M. Ojala (University of Helsinki, Helsinki, Finland) and were grown in EGM-2MV medium (Lonza, Walkersville, MD, USA).

Techniques: Infection, Control, Expressing, Western Blot, Transfection, Virus, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Toll-Like Receptor 4 Decoy, TOY, Attenuates Gram-Negative Bacterial Sepsis

doi: 10.1371/journal.pone.0007403

Figure Lengend Snippet: LEC (primary lymphatic microvascular endothelial cells derived from human adult dermis) were purchased from Cambrex Inc. (East Rutherford, NJ) and maintained in endothelial cell basal medium-2 with growth supplements (EBM-2 MV). Passage 4–6 LEC were incubated in EBM-2 MV containing 1 % FBS for 8 hr and then with 1 µg/ml of Fc, TFE, TOY3, or TOY8 for 15 min, and then the LEC were treated with LPS (500 ng/ml) for 30 min. (A) For determination of NF-κB activation, nuclear translocalization of p65 (a subunit of NF-κB) was analyzed by immunostaining (green). Nuclei were counterstained with DAPI (blue). Arrows indicate nuclear translocalization of p65. Scale bars, 100 µm. (B) Cells positive for p65 intranuclear staining (white arrows) were counted among 100 cells arbitrarily chosen in 4 different regions, and the values presented as a percentage of the total cell number. Bars represent means ± S.D. ( n = 4). *, P <0.05 versus Fc. (C) Primary cultured macrophages from mouse peritoneal cavity were pre-treated with 1.0 µg/ml of Fc, TOY3, or TOY8 for 30 min, and then were treated with LPS (100 ng/ml) for 4 hr. Culture media were sampled, and levels of TNF-α were measured. Bars represent means ± S.D. ( n = 5). *, P <0.05 versus Fc.

Article Snippet: Briefly, LEC (primary lymphatic microvascular endothelial cells derived from human adult dermis) were purchased from Cambrex Inc. (East Rutherford) and maintained in endothelial cell basal medium-2 with growth supplements (EBM-2 MV).

Techniques: Derivative Assay, Incubation, Activation Assay, Immunostaining, Staining, Cell Culture