Microscope camera system

Immunohistochemical detection of macrophages using CD68 (Neomarker Microm, Francheville, France) was performed with the avidin–biotin peroxidase (ABC) method. Processed slide images were acquired by a microscope-camera system at ×40 magnification (Nikon, France).
Slides of abdominal adipose tissue were also stained with picrosirius red. Fibrosis analysis was performed using Alphalys platform (histolab software, Plaisir, France) at ×100 magnification with constant color thresholds.
Adipose tissue samples were fixed overnight at 4 °C in 4% paraformaldehyde and processed for standard paraffin embedding. Sections 5 μm thick were stained as described below and examined under a Zeiss 20 Axiostar Plus microscope (Zeiss, Germany). Digital images were captured by a camera (triCCD, Sony, France). Adipocyte diameters were measured using perfectimage Software (Claravision, France).
Two sections per sample were analyzed. The two sections were spaced by 25 micrometers, that is, five sections spacing. The observation was made blindly by two independent observers.

In vitro tube formation was evaluated using the Angiogenesis Assay Kit (PromoCell, Heidelberg, Germany; PK-CA577-K905) according to the manufacturer’s instructions. Briefly, 50 μL/well ECM solution was added to prechilled 96-well plates and incubated for 1 h at 37 °C to form a gel. HAMON or ISO-HAS-B cells were transfected with control or NECTIN4 siRNA for 48 h, seeded into the ECM-treated wells at a density of 1–2 × 10⁴ cells/well, and incubated for 24 h at 37 °C in 5% CO₂. To inhibit PI3K/Akt, Src, or VEGFR2, or to activate Src, cells were treated with DMSO (0.1%), LY294002 (10 μM), dasatinib (100 nM), cabozantinib (10 μM), or MLR-1023 (1 μM) respectively for 48 h at 37 °C in 5% CO₂, seeded into the ECM-treated wells, and then incubated for 24 h at 37 °C in 5% CO₂. HUVEC were used as a positive control because, in this assay system, they form tubular structures. After incubation, cells were observed under a microscope and images were captured using a microscope camera system (Nikon). The number of tubules, tubule length, and the number of junctions were measured using ImageJ software (National Institutes of Health, Bethesda, MD).

HUVEC (Takara Bio, Kusatsu, Japan; C-12203), HDMEC (Takara Bio; C-12212), HDBEC (Takara Bio; C-12211), and HAMON human angiosarcoma cells^{44 (link),45 (link)} (kindly provided by Riichiro Abe, Division of Dermatology, Niigata University) were cultured in Endothelial Cell Growth Medium 2 (Takara Bio; C-22111), containing 2% fetal calf serum, 5 ng/mL human epidermal growth factor, 0.2 μg/mL hydrocortisone, 22.5 μg/mL heparin, 20 ng/mL R3 insulin-like growth factor-1, 1 μg/mL ascorbic acid, 10 ng/mL human basic fibroblast growth factor, and 0.5 ng/mL VEGF. ISO-HAS-B human angiosarcoma cells (Resource Center for Biomedical Research, Institute of Development, Aging, and Cancer, Tohoku University) were cultured in DMEM (Sigma Aldrich; D6429) supplemented with 10% FBS. Cells were passaged at confluence using a Detach Kit (Takara Bio; C-41210) according to the manufacturer’s instructions, and the medium was changed every 2–3 days. The CycleavePCR Mycoplasma Detection Kit (Takara Bio; CY232) was used to test for mycoplasma contamination, and cells were confirmed to be mycoplasma free. Cell morphology was observed under a microscope and pictures were captured using a microscope camera system (Nikon).