P3X63Ag8U.1 (P3U1), Chinese hamster ovary (CHO)-K1, LN229, and NMuMG (a mouse mammary gland epithelial cell) were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). Lewis lung carcinoma was obtained from the Japanese Collection of Research Bioresources (JCRB; Osaka, Japan).
The pCAG-zeo_ssnPA-mEGFR and pCAG-zeo_MAP-mEGFR plasmids were transfected into LN229 and CHO-K1 cells, respectively. The stable transfectants were generated as described previously [40 (link),41 (link)].
CHO-K1, mEGFR-overexpressed CHO-K1 (CHO/mEGFR), Lewis lung carcinoma, and P3U1 were cultured in an RPMI-1640 medium (Nacalai Tesque Inc., Kyoto, Japan), with 10% fetal bovine serum (FBS; Thermo Fisher Scientific Inc., Waltham, MA, USA). A cocktail of 100 units/mL of penicillin, 100 μg/mL of streptomycin, and 0.25 μg/mL of amphotericin B (Nacalai Tesque Inc.) was added to the medium. LN229, mEGFR-overexpressed LN229 (LN229/mEGFR), and NMuMG were cultured in DMEM (Nacalai Tesque Inc.), supplemented as indicated above. For NMuMG cells, 10 μg/mL of insulin (Sigma-Aldrich Corp., St. Louis, MO, USA) was further added. All cells were cultured using a humidified incubator at 37 °C, in an atmosphere of 5% CO2 and 95% air.
The pCAG-zeo_ssnPA-mEGFR and pCAG-zeo_MAP-mEGFR plasmids were transfected into LN229 and CHO-K1 cells, respectively. The stable transfectants were generated as described previously [40 (link),41 (link)].
CHO-K1, mEGFR-overexpressed CHO-K1 (CHO/mEGFR), Lewis lung carcinoma, and P3U1 were cultured in an RPMI-1640 medium (Nacalai Tesque Inc., Kyoto, Japan), with 10% fetal bovine serum (FBS; Thermo Fisher Scientific Inc., Waltham, MA, USA). A cocktail of 100 units/mL of penicillin, 100 μg/mL of streptomycin, and 0.25 μg/mL of amphotericin B (Nacalai Tesque Inc.) was added to the medium. LN229, mEGFR-overexpressed LN229 (LN229/mEGFR), and NMuMG were cultured in DMEM (Nacalai Tesque Inc.), supplemented as indicated above. For NMuMG cells, 10 μg/mL of insulin (Sigma-Aldrich Corp., St. Louis, MO, USA) was further added. All cells were cultured using a humidified incubator at 37 °C, in an atmosphere of 5% CO2 and 95% air.
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