Cellect

Normal human epidermal keratinocytes (NHEKs) (FC-0025, Lifeline Cell Technology, Frederick, USA; purchased from Kurabo, Osaka, Japan) were cultured in Humedia-KG2 medium supplemented with growth factors (Kurabo). The cells were exposed to 2 mM CaCl₂ for 2 days to induce differentiation. The human keratinocyte cell line HaCaT (gift from Dr. Manabe) has been maintained for more than ten years in DMEM/Ham’s F12 (DH) medium (Fujifilm-Wako, Osaka, Japan) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (CELLect, MP Biomedicals, Illkirch, France), 50 U/mL penicillin, and 50 μg/mL streptomycin (Meiji Seika Pharma, Osaka, Japan) (DH10). These cells, their transgenic derivatives, and Cos7 cells were cultured in DH10 medium. For certain cultures, the cells were treated with 5 μM MG132 (Abcam, Cambridge, UK) for 4 or 7 h. For induction of tight junction (TJ) formation, cells were treated with 10 mM CaCl₂ and 40 μM the c-Jun N-terminal kinase (JNK) inhibitor SP600125 (Sigma‒Aldrich, St. Louis, USA) for 24 h.

Human cervical adenocarcinoma cells (HeLa) and lung adenocarcinoma cells (NCI-H1975) were purchased from the RIKEN Cell Bank (Tsukuba, Japan) and American Type Culture Collection (Manassas, VA), respectively. These cells were grown in the RPMI-1640 medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (CELLect®, MP Biomedicals, Santa Ana, CA) and 100 units/mL of penicillin-streptomycin (MP Biomedicals). The cells were maintained at 37°C in 5% CO₂.