FHC, a human colon epithelial normal cells, were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and cultured according to the recommended procedure by the supplier. HEK293T and CRC cells (Caco-2, COLO205, SW480, CW-2, LoVo, SW48 and HCT116) were obtained as reported previously (Inaguma et al., 2017 (link); Inoue et al., 2020 (link)). CRC cells were maintained in Dulbecco's Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).
The lentivirus vectors for PBK and the control LacZ were constructed using the CSII-EF-MCS plasmid, which was kindly provided by Dr. H. Miyoshi (RIKEN BioResource Center, Tsukuba, Japan). The pcDNA3.1 vector (Invitrogen) was used to express PBK, CDH1cyt (CDH1 cytoplasmic domain, R733 to D882) or firefly luciferase (Luc2CP). The CDH1cyt mutants (CDH1cyt/S840,846,847A and CDH1cyt/S840,846,847D) were generated by PCR-based mutagenesis. The pGEX-5X-1 vector was used for the synthesis and purification of glutathione S-transferase (GST)-tagged proteins.
OTS514, a selective PBK inhibitor, was purchased from Selleck Biotech (Tokyo, Japan) and used to treat cells for 24–72 h. Colchicine was obtained from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan) and added at a concentration of 1 × 10–7 M for 8 h. MG132, a proteasome inhibitor, was from Selleck Biotech (Tokyo, Japan) and applied at 1 µM for 8 h.
The lentivirus vectors for PBK and the control LacZ were constructed using the CSII-EF-MCS plasmid, which was kindly provided by Dr. H. Miyoshi (RIKEN BioResource Center, Tsukuba, Japan). The pcDNA3.1 vector (Invitrogen) was used to express PBK, CDH1cyt (CDH1 cytoplasmic domain, R733 to D882) or firefly luciferase (Luc2CP). The CDH1cyt mutants (CDH1cyt/S840,846,847A and CDH1cyt/S840,846,847D) were generated by PCR-based mutagenesis. The pGEX-5X-1 vector was used for the synthesis and purification of glutathione S-transferase (GST)-tagged proteins.
OTS514, a selective PBK inhibitor, was purchased from Selleck Biotech (Tokyo, Japan) and used to treat cells for 24–72 h. Colchicine was obtained from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan) and added at a concentration of 1 × 10–7 M for 8 h. MG132, a proteasome inhibitor, was from Selleck Biotech (Tokyo, Japan) and applied at 1 µM for 8 h.
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