Hdbec

CHO cells were infected at a MOI of 100 and used to produce adenovirus‐conditioned media for 3 days. Normal human dermal fibroblast cells (NHDF, Promocell) were seeded onto coverslips for 3 days. Human dermal blood endothelial cells (HDBECs, Promocell) were then seeded on top of the fibroblasts and allowed to settle overnight. The addition of fibroblasts allowed for a continuously renewing layer of fibronectin and collagen to be synthesized, which supported spontaneous organization of ECs into tubule‐like structures.15 The cell culture media was then replaced with half volume of fresh media and half volume adenovirus‐conditioned media and changed every 2 days for 2 weeks, thus enabling us to test the functionality of the proteins produced via adenoviral‐mediated infection. After 2 weeks, the media was removed, and the cells were fixed with ethanol, stained with polyclonal rabbit anti‐VE‐Cadherin (5 μg/mL, ab33168, Abcam), and counterstained with DAPI. z‐stack images of 5 sections containing tubes from each coverslip were acquired, and image analysis was undertaken blinded using ImageJ.16 We counted number of endothelial tubes, measured tube segment length, and expressed sprout point and branch point number per unit area.

HDBECs were purchased from PromoCell (Heidelberg, Germany) and cultured in endothelial cell growth medium MV (PromoCell) at 37 °C in 5% CO₂ until reaching 70–80% confluency. For the IgG endocytosis assay, cells were incubated in the presence of A594-conjugated human IgG (Cat. 009-580-003, 10 μg ml⁻¹; Jackson ImmunoResearch) for 1 h. To block dynamin function, cells were treated with dynasore (100 μg ml⁻¹; Santa Cruz Biotechnology, Dallas, TX) for 30 min. To block caveolae-mediated endocytosis, cells were cultured with nystatin (50 μg ml⁻¹; Sigma Aldrich) for 30 min. To evaluate the quantity of engulfed IgG in each cell, the cytoplasmic area of cells in a high-power field (×40) was manually circumscribed using ImageJ^{58 (link)}, and the MFI of the signals from A594-IgG was measured. ΔA594-IgG was calculated by subtracting the background MFI.

Human umbilical vein ECs (HUVECs) and human dermal blood ECs (HDBECs) were purchased from PromoCell (Heidelberg, Germany). Cells were grown on gelatin-coated plates as previously described [18 (link), 26 (link)]. For spreading analyses, cells were detached from the culture plate by using enzyme-free cell dissociation buffer (Thermo Fisher Scientific, Waltham, MA, USA), plated on gelatin-coated coverslips, and allowed to spread for different times. Cells at specific phases of spreading were fixed, labeled, and imaged by microscopy. Transient transfection experiments were performed by electroporation as previously described [12 (link)].

GL261 glioma cells (a gift from Geza Safrany, National Research Institute for Radiobiology and Radiohygiene, Budapest, Hungary) were cultured in DMEM (Life Technologies) supplemented with 10% FBS (Sigma-Aldrich) at 37°C and 5% CO₂/95% air in a humidified chamber.
Human dermal microvascular endothelial cells (HDMECs) or human dermal blood endothelial cells (HDBECs) (PromoCell) were cultured in gelatin-coated culture dishes in Endothelial Cell Basal Medium with full supplements (PromoCell, EBM-MV2) at 37°C and 5% CO₂/95% air in a humidified chamber.
Primary mouse brain endothelial cells were isolated from 12-week-old C57BL/6 WT or CD93^–/– mice as previously described (48 (link)).
Mycoplasma test was routinely performed in all cell cultures used in this study.

Mutated over-active CARD14 (E138A and G117S), as well as wild-type CARD14 expression plasmids (full-length (fl) and short (sh)) (REF) were described elsewhere [2] (link). HDBECs (PromoCell, Heidelberg, Germany) were cultured as previously described [27] (link). HDBECs were transfected using PromoFectin-HUVEC according to manufactures recommendations. In brief, HDBECs were seeded into a 24 well plate and allowed to reach 50%-70% confluency. PromoFectin-HUVEC and expression plasmid DNA were diluted in serum-free Endothelial Cell Basal Medium (ECBM) (PromoCell). 2 ul of PromoFectin-HUVEC was used per 1 ug of plasmid DNA, and allowed to incubate at room temperature for 30 minutes. HDBECs were washed with ECBM. Transfections took place in 500 ul of serum-free medium for 6 hours, after which media was replaced with EC media containing serum. Using this method, transfection efficiency of HDBEC was 60–65%, as determined by a GFP-expression plasmid and subsequent flow cytometry analysis. For stimulation with TNFα (25 ng/mL), cells were allowed to rest for 3 hours after transfection. RNA and supernatant were collected 24 hours post-transfection.