Huvecs

HUVECs were purchased from Kurabo (Osaka, Japan) and seeded in plastic plates precoated with type I collagen (Asahi Techno Glass, Nagoya, Japan) and were maintained in endothelial cell growth medium (Promo cell, Heidelberg, Germany) supplemented with 0.5 μg/mL fungizone, 0.25 μg/mL amphotericin B, 100 μg/mL streptomycin, and 100 U/mL penicillin (Life Technologies, Carlsbad, CA, USA).

Human colon cancer DLD-1 (Cat# JCRB9094), WiDr (Cat# JCRB0224), and COLO201 (Cat# JCRB0226) cells were obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank (Osaka, Japan), while SW480 (SW-480) (ATCC^® CCL-228^TM) cells were obtained from American Type Culture Collection (Manassas, VA). HUVECs were obtained from KURABO (Osaka, Japan). The colon cancer cells were maintained in RPMI-1640 (Sigma-Aldrich, St Louis, MO) containing 10% (v/v) heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA), while the HUVECs were maintained in complete medium (Humedia-EG2; KURABO). All cell lines were cultured under an atmosphere of 95% air and 5% CO₂ at 37 °C. The Trypan blue exclusion test was used to determine the number of viable cells in a cell suspension, from which the cell proliferation ratio was calculated.

HUVECs were purchased from Kurabo and were maintained in HuMedia-EG2 with a growth additive set. HEK293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Nissui, Tokyo, Japan) containing 10% fetal calf serum and antibiotics. HUVECs and HEK293 cells were transfected by using the NEON electroporation system (Invitrogen) and Lipofectamine 2000 (Invitrogen), respectively.

The human prostate cancer LNCaP cell line was purchased from the American Type Culture Collection (ATCC) and maintained in Dulbecco's-modified Eagle's medium (DMEM) (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal bovine serum (FBS) containing antibiotics (penicillin, 50 IU/mL; streptomycin, 50 μg/mL) at 37°C in 5% CO₂. LNCaP-SF cells were established after long-term subculture of parental LNCaP cells in DMEM and 5% charcoal-stripped fetal calf serum (CCS) [26 (link)] and maintained in DMEM supplemented with 5% CCS and antibiotics at 37°C in 5% CO₂. The human prostate cancer 22Rv1 cell line was purchased from ATCC and maintained in Roswell Park Memorial Institute 1640 medium (RPMI 1640; Gibco) supplemented with 10% heat-inactivated FBS containing antibiotics (penicillin, 50 IU/mL; streptomycin, 50 μg/mL) at 37°C and 5% CO₂. Human umbilical vein endothelial cells (HUVECs) were purchased from KURABO (Cat#: KE-4109P10, Strain No. 04609, Osaka, Japan) and maintained in EGM-2 bullet kit medium (CC-3162, Lonza, Walkersville, MD).

Wild-type (WT) C57BL/6 mice were purchased from Japan SLC (Shizuoka, Japan). Apelin-knockout (KO) mice on the C57BL/6 background were generated as described previously^{17 (link)}. APJ-knockout (KO) mice were gifts from Prof. Fukamizu^{37 (link)}. All mice were used at the age of 8–10 weeks. Animals were housed in environmentally-controlled rooms in the animal experimentation facility at Osaka University. All experiments were performed in compliance with the laws and institutional guidelines of Osaka University Committee for Animal and Recombinant DNA Experiments. This study was approved by Osaka University Research Ethics Committee (Approval number 4062). This research was carried out in compliance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.
MC38 (mouse colon carcinoma cell line) and LLC (mouse Lewis lung carcinoma cell line) purchased from the Riken cell bank (Tsukuba, Japan) were maintained in DMEM (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS, Sigma) and 1% penicillin/streptomycin (100 U/ml, PS; Life Technologies, Tokyo, Japan). HUVECs purchased from Kurabo (Osaka, Japan) were cultured in Humedia EG2 (Kurabo). They were starved and then stimulated with VEGF-A (20 ng/ml, PeproTech, Rocky Hill, NJ, USA) for 24 h after which [Pry¹]Apelin-13 (1 µg/ml) was added for 6 h.

The expression of glycan epitopes on the cell surface was analyzed using an RF-500 flow cytometer (Sysmex, Tokyo, Japan). HUVECs purchased from KURABO (Osaka, Japan) were maintained in HuMedia-EG2 (KURABO) at 37 °C in a humidified atmosphere containing 5% CO₂. The HUVECs have been obtained under proper informed consent and adheres to the Declaration of Helsinki, the Human Tissue Act (UK), Title 21 of the Code of Federal Regulations (USA), and HIPAA regulations relative to obtaining and handling human tissue for research use. 1 × 10⁶ cells were seeded onto a culture dish (100 mm in diameter) and incubated overnight. After the incubation, cells were harvested using 5 mM ethylenediaminetetraacetic acid in PBS, and suspended in 200 μl of cold PBS. The suspensions were incubated with 0.2 μg of FR9, AFR45, or PA5 monoclonal IgM on ice, then sequentially labeled with Alexa 488-conjugated anti-mouse IgM antibody (Thermo Fisher Scientific).