Cho k1

CHO-K1 and a canine osteosarcoma cell line D-17 were purchased from the American Type Culture Collection (Manassas, VA, USA). Stable dog EGFR-overexpressed CHO-K1 (CHO/dEGFR) [26 (link)] and dog HER2-overexpressed CHO-K1 (CHO/dHER2) [27 (link)] were established as described previously. CHO-K1, CHO/dEGFR, and CHO/dHER2 were cultured in RPMI-1640 medium (Nacalai Tesque, Inc., Kyoto, Japan), containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 μg/mL streptomycin, 100 units/mL of penicillin, and 0.25 μg/mL amphotericin B (Nacalai Tesque, Inc.). D-17 was cultured in MEM medium (Nacalai Tesque, Inc.), supplemented with 10% FBS, 1 mM of sodium pyruvate, 100 units/mL of penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B.

The human OSCC cell line (HSC-3) and the human gastric cancer cell lines (MKN45 and NUGC-4) were obtained from the Japanese Collection of Research Bioresources (Osaka, Japan). The human pancreatic cancer cell line (PANC-1) was obtained from the Cell Resource Center for Biomedical Research Institute of Development, Aging, and Cancer at Tohoku University (Sendai, Japan). Chinese hamster ovary (CHO)-K1 and P3X63Ag8U.1 (P3U1; a mouse multiple myeloma) cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). HSC-3 was cultured in DMEM medium (Nacalai Tesque, Inc., Kyoto, Japan), supplemented with 100 μg/mL streptomycin, 100 U/mL penicillin, and 0.25 μg/mL amphotericin B (Nacalai Tesque, Inc.), and 10% (v/v) heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The cell lines (MKN45, NUGC-4, PANC-1, CHO-K1, and P3U1) were cultured in RPMI-1640 medium (Nacalai Tesque, Inc.), supplemented as indicated above. All cells were cultured using a humidified incubator at 37 °C, in an atmosphere of 5% CO₂ and 95% air.

Chinese hamster ovary (CHO)-K1, a human glioblastoma cell line (LN229), and mouse multiple myeloma P3X63Ag8U.1 (P3U1) cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The human OSCC cell line, HSC-3, was obtained from the Japanese Collection of Research Bioresources (Osaka, Japan). CHO-K1 and P3U1 were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Nacalai Tesque, Inc., Kyoto, Japan), supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 0.25 μg/mL amphotericin B (Nacalai Tesque, Inc.), and 10% heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific, Inc., Waltham, MA, USA).
LN229 and HSC-3 were cultured in Dulbecco’s modified Eagle medium (DMEM) (Nacalai Tesque, Inc.), supplemented with 10% (v/v) FBS, 100 U/mL of penicillin (Nacalai Tesque, Inc.), 100 μg/mL streptomycin (Nacalai Tesque, Inc.), and 0.25 μg/mL amphotericin B (Nacalai Tesque, Inc.). LN229/CD44ec was cultured in the presence of 0.5 mg/mL of G418 (Nacalai Tesque, Inc.).
All the cells were grown in a humidified incubator at 37 °C with 5% CO₂.

LN229, A431, Chinese hamster ovary (CHO)-K1, HEK-293T, MCF-10A, Met-5A, and P3U1 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA). HSC-2 and HSC-3 were obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank (Osaka, Japan). LN229/EGFR and CHO/EGFR were produced by transfecting pCAG/PA-EGFR-RAP-MAP^{(12 (link))} into LN229 and CHO-K1 cells using Neon transfection system (Thermo Fisher Scientific, Inc., Waltham, MA) and Lipofectamine LTX (Thermo Fisher Scientific, Inc.), respectively. A few days after transfection, PA tag-positive cells^{(13 (link))} were sorted using a cell sorter (SH800; Sony Corp., Tokyo, Japan).
CHO-K1, CHO/EGFR, and P3U1 cell lines were cultured in RPMI 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan), and LN229, LN229/EGFR, A431, HSC-2, HSC-3, HEK-293T, MCF-10A, and Met-5A cell lines were cultured in Dulbecco's modified Eagle's medium (Nacalai Tesque, Inc.), supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, Inc.), 100 units/mL of penicillin, 100 μg/mL of streptomycin, and 25 μg/mL of amphotericin B (Nacalai Tesque, Inc.) at 37°C in a humidified atmosphere containing 5% CO₂ and 95% air.

CHO-K1, a canine osteosarcoma cell line (D-17 [17 (link)]), and a canine fibroblast cell line (A-72 [18 (link)]) were obtained from the American Type Culture Collection (Manassas, VA, USA). CHO/dEGFR was established in our previous study [16 (link)]. CHO-K1 and CHO/dEGFR were cultured in RPMI medium (Nacalai Tesque, Inc., Kyoto, Japan), D-17 in Minimum Essential Medium (Nacalai Tesque, Inc., Kyoto, Japan), and A-72 in Dulbecco’s Modified Eagle Medium (DMEM; Nacalai Tesque, Inc., Kyoto, Japan). Those media were supplemented with 10% heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific Inc., Waltham, MA, USA), 1 mM of sodium pyruvate, 100 units/mL of penicillin, 100 μg/mL of streptomycin, and 0.25 μg/mL of amphotericin B (Nacalai Tesque, Inc., Kyoto, Japan). The cells were cultured at 37 °C in a humidified atmosphere containing 5% CO₂. The negativity for mycoplasma infection was confirmed using the TaKaRa Mycoplasma Detection Set (Takara Bio, Shiga, Japan).

COLO205 (a human colorectal cancer cell line) was obtained from the Cell Resource Center for Biomedical Research Institute of Development, Aging and Cancer at Tohoku University (Sendai, Japan). HSC-3 (a human OSCC cell line) and LN229 (a human glioblastoma cell line) were obtained from the Japanese Collection of Research Bioresources (Osaka, Japan). P3X63Ag8U.1 (P3U1: a mouse multiple myeloma) and CHO-K1 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). HSC-3 and LN229 were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Nacalai Tesque, Inc., Kyoto, Japan), supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 U/mL of penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B. CHO-K1, COLO205, and P3U1 were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Nacalai Tesque, Inc.) supplemented with 10% FBS and antibiotics, as indicated above. All the cells were grown in a humidified incubator at 37 °C with 5% CO₂.

HSC-2 (oral squamous carcinoma from oral cavity), HSC-3 (oral squamous carcinoma cell line from tongue with high metastatic potential), HSC-4 (oral squamous carcinoma cell line from tongue), Ca9-22 (oral squamous carcinoma from gingiva), HO-1-u-1 (oral squamous carcinoma from mouth floor), and SAS (oral squamous carcinoma cell line from tongue) were obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). CHO-K1 was obtained from the American Type Culture Collection (ATCC, Manassas, VA). CHO/hPODXL was produced in our previous study [17 (link)]. The CHO-S cell line was purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). PDIS-5 (core fucose KO CHO-S) cells were established previously [48 (link)]. SAS/hPODXL-KO cells were produced using CRISPR/Cas9 plasmids (Target ID: HS0000056763) targeting human PODXL (Sigma-Aldrich Corp., St. Louis, MO, USA). HSC-2, HSC-3, HSC-4, Ca9-22, HO-1-u-1, SAS and SAS/hPODXL-KO cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Nacalai Tesque, Kyoto, Japan), and CHO-K1 and CHO/hPODXL were cultured in RPMI 1640 medium (Nacalai Tesque), supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific Inc.), 100 units/mL penicillin, 100 μg/mL streptomycin, and 25 μg/mL amphotericin B (Nacalai Tesque) at 37°C in a humidified atmosphere containing 5% CO₂ and 95% air.