Cho k1

Parental Chinese hamster ovary cells (CHO-K1, ATCC: CRL-11268) were cultivated in F-12 Nut Mix (Ham) medium (Thermo Fisher Scientific Inc., ref. 21765-029) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) (Euroclone S.p.A., cat. no. ECS0180L) and 1% (v/v) penicillin/streptomycin solution (Lonza, cat. no. DE17-602E). Heat inactivation of FBS was performed by incubation in 56°C water bath for 30 min.
Human embryonic kidney cells (HEK-293T, ATCC: CRL-11268) and HeLa cells (HeLa, ATCC: CCL-2) were cultivated in Dulbecco's modified Eagle medium (DMEM; Thermo Fisher Scientific Inc., ref. 11960-044) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) (Euroclone S.p.A., cat. no. ECS0180L), 1% (v/v) L-Glutamine (Lonza, cat. no. 17-605E), and 1% (v/v) penicillin/streptomycin solution (Lonza, cat. no. DE17-602E).
All cell lines were cultivated in 75 mL cell culture flasks at 37°C in a humidified atmosphere containing 5% CO₂. Subculture was performed every 3-4 days, at about 80% confluence. After 2x wash using Dulbecco's phosphate-buffered saline (DPBS) (Thermo Fisher Scientific Inc., ref. 14040-091), cells were detached from the surface of the culture flask by using a TrypLE Express reagent (Thermo Fisher Scientific Inc., ref. 12605-010). Viable cell numbers were determined using a TC20 Automated Cell Counter (Bio-Rad Laboratories, Inc.).

Chinese hamster ovary (CHO)-K1 was obtained from the American Type Culture Collection (Manassas, VA). The wPDPN mutation plasmids containing the RIEDL tag were transfected into CHO-K1 cells using Lipofectamine LTX (Thermo Fisher Scientific, Inc., Waltham, MA). Transiently transfected cells with deletion mutants or point mutants were cultured in RPMI 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan), supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Thermo Fisher Scientific, Inc.), 100 U/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 of 5% CO₂ and 95% air.

HUVECs were purchased from PromoCell. The cells were maintained in EGM-2 basal medium with SupplementPack (complete EGM, PromoCell) at 37 °C in a humidified atmosphere and 5% CO₂. Human cervical cancer HeLa 229, human lung fibroblast MRC-5, and chinse hamster ovary CHO-K1 cells were obtained from Japanese Collection of Research Bioresources cell bank. The cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS, Biowest) (for HeLa 229 and MRC-5; Thermo Scientific) or Ham’s F-12 medium/10% FBS (for CHO-K1; Thermo Scientific) at 37 °C in a humidified atmosphere and 5% CO₂.