Cho k1

BT-474, SK-BR-3, MCF 10A, and CHO-K1 cell lines were sourced from the American Type Culture Collection (Manassas, VA, USA). 293FT and HaCaT cells were obtained from Thermo Fisher Scientific Inc. (Thermo, Waltham, MA, USA) and Cell Lines Service GmbH (Eppelheim, Germany), respectively. These cells were maintained as described previously [41 (link)].

Cryopreserved primary HUVECs (Cat# PCS-100-013), CHO-K1 (Cat# CCL-61) cells and pgsD-677 (CHO D-677, Cat# CRL-2244) cells were purchased from ATCC, and HDMECs were purchased from LONZA (Cat# CC-2543). Both HUVECs and HDMECs were cultured in complete vascular cell growth medium (Vascular Cell Basal Medium: (Cat# PCS-100-030), supplemented with Endothelial Cell Growth Kit-VEGF (Cat# PCS-100-041; ATCC) to yield final concentrations of rh VEGF: 5 ng/mL, rh EGF: 5 ng/mL, rh FGF basic: 5 ng/mL, rh IGF-1: 15 ng/mL, L-glutamine: 10 mM, heparin: 0.75 units/mL, hydrocortisone: 1 µg/mL, ascorbic acid: 50 µg/mL, fetal bovine serum: 2%, also containing penicillin-streptomycin (100 U/mL, Cat# 15140122; Gibco) and maintained at 37 °C in 5% CO₂. To subculture, HUVECs were washed once with PBS, treated with 0.05% trypsin-EDTA (Cat# 25300054; Thermo Fisher Scientific) for 90 s and were used until passage three. After purification by FACS (see below), human primary CD4⁺ T cells were cultured overnight in RPMI-10 medium (RPMI 1640, Cat# 11875119; Gibco), penicillin-streptomycin (100 U/ml, Cat# 15140122; Gibco) and HEPES (10 mM, Cat# 15630080; Gibco) containing 10% FBS (Cat# 100-500; Gemini Bio-product) at 37 °C in 5% CO₂ before using in experiments. CHO-K1 and D-677 cells were cultured in F12K medium (30-2004, ATCC) supplemented with 10% fetal bovine serum.

Cell lines, including LN229, Chinese hamster ovary (CHO)–K1, and P3X63Ag8U.1 (P3U1) cells were obtained from the American Type Culture Collection (Manassas, VA, USA). DLD-1 cells were obtained from the Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University (Miyagi, Japan). The expression plasmid of EphB6 (pCMV6neoEphB6-Myc-DDK, Catalog No.: RC229404, Accession No.: NM_004445, OriGene Technologies, Inc. Rockville, MD, USA) was transfected into cell lines using the Neon transfection system (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Subsequently, LN229 and CHO–K1, which stably overexpressed EphB6 with C-terminal Myc-DDK tags (hereafter described as LN229/EphB6 and CHO/EphB6, respectively) were stained with an anti-EphB6 mAb (clone T49-25; BioLegend, San Diego, CA, USA) and sorted using the SH800 cell sorter (Sony corp., Tokyo, Japan), followed by cultivation in a medium containing 0.5 mg/mL of G418 (Nacalai Tesque, Inc., Kyoto, Japan).
The complementary DNAs (cDNAs) of other Eph receptors, including EphA1 (Catalog No.: RC213689, Accession No.: NM_005232), EphA4 (Catalog No.: RC211230, Accession No.: NM_004438), EphA5 (Catalog No.: RC213206, Accession No.: NM_004439), EphA6 (Catalog No.: RC223510, Accession No.: NM_001080448), EphA7 (Catalog No.: RC226293, Accession No.: NM_004440), EphA8 (Accession No. NM_020526; Catalog No.: RC220352), EphA10 (Catalog No.: RC218374, Accession No.: NM_001099439), EphB1 (Catalog No.: RC214301, Accession No.: NM_004441), EphB2 (Catalog No.: RC223882, Accession No.: NM_004442) were purchased from OriGene Technologies (Rockville, MD, USA), Inc. EphA2 (Catalog No.: HGY095959, Accession No.: NM_004431), EphA3 (Catalog No.: HGY053437, Accession No.: NM_005233), and EphB3 (Catalog No.: HGX039581, Accession No.: NM_004443) cDNAs were purchased from RIKEN DNA Bank (Ibaraki, Japan).
EphA2 and EphB3 cDNAs were cloned into a pCAGzeo vector [FUJIFILM Wako Pure Chemical Corporation (Wako), Osaka, Japan]. EphA1 cDNA was cloned into a pCAGzeo-ssnPA vector. EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA10, and EphB1 cDNA were cloned into a pCAGzeo_ssnPA16 vector.
The plasmids were also transfected into CHO–K1 cells and stable transfectants were established by staining with an anti-EphA2 mAb (clone SHM16; BioLegend), an anti-EphB2 mAb (clone 2H9; BD Bioscience, Franklin Lakes, NJ, USA), an anti-EphB3 mAb (clone 647354; R&D Systems Inc., Minneapolis, MN, USA), and an anti-PA tag [46 (link)] mAb (clone NZ-1 for EphA1, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA10, and EphB1), and sorted using SH800. After sorting, cultivation in a medium containing 0.5 mg/mL of Zeocin (InvivoGen, San Diego, CA, USA) or 0.5 mg/mL of G418 was progressed. These Eph receptors-overexpressed CHO–K1 (e.g., CHO/EphA1) clones were finally established. CHO/PA16-EphB4 was previously described [45 (link)].
CHO–K1, P3U1, Eph receptor-overexpressed CHO–K1, and DLD-1 cells were also cultured in a Roswell Park Memorial Institute (RPMI)-1640 medium (Nacalai Tesque, Inc.) that was supplemented with 10 % heat-inactivated fetal bovine serum (FBS, Thermo Fisher Scientific Inc.), 100 units/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B (Nacalai Tesque, Inc.). LN229 and LN229/EphB6 were cultured in a Dulbecco's Modified Eagle Medium (DMEM) (Nacalai Tesque, Inc.) that was supplemented with 10 % heat-inactivated FBS (Thermo Fisher Scientific Inc.), 100 units/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B (Nacalai Tesque, Inc.). Then, cells were cultured in a humidified CO₂ incubator with 5 % CO₂ and 95 % air at 37 °C.