D4476

HAM2 and HAM3 ameloblastoma cell lines were established from a human ameloblastoma tissue, as described previously21 (link). HAM cell lines were maintained at 37 °C in 5% CO₂ in keratinocyte-SFM medium (K-SFM; Life Technologies, Carlsbad, CA, USA). DLD1 colorectal cancer cells were purchased from ATCC (Manassas, VA, USA) and cultured at 37 °C in 5% CO₂ in DMEM medium (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 5% FBS (AusGeneX, Molendinar, QLD, Australia). MCF10A mammary gland cells were obtained from the Barbara Ann Karmanos Cancer Institute Cell Line Resource (Detroit, MI, USA) and cultured at 37 °C in 5% CO₂ in IMDM medium (Life Technologies) supplemented with 10% FBS (Life Technologies). Plasmid DNAs and siRNAs were transfected using Lipofectamine 3000 and Lipofectamine RNAiMAX, respectively (Life Technologies). The transfection of HAM cell lines was performed in IMDM medium supplemented with 10% FBS because K-SFM medium was not suitable for the transfection reaction. D4476, a CK-1 inhibitor, was used at a concentration of 100 μM (Abcam, Cambridge, UK).

IL-6 and IPTG were from Sigma-Aldrich (Italy); BZ and Lena from Selleck chemicals (USA); D4476 (4-[4-(2,3-Dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide) from abcam (UK); Z-VAD-FMK from Enzo Life Science (UK).

HCT116, RKO, WiDr, and Caco-2 colorectal cancer cells and an MAb104 hybridoma were purchased from ATCC (Manassas, VA, USA). DLD1 cells were kindly provided by Dr. Tagawa (Chiba Cancer Center Research Institute, Chiba, Japan). These cell lines, except for the MAb104 hybridoma, were maintained at 37 °C in 5% CO₂ in IMDM (Life Technologies, Carlsbad, CA, USA) or DMEM (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 5 or 10% FBS. The MAb104 hybridoma was cultured in GIT medium (Nihon Pharmaceutical Co., LTD., Tokyo, Japan). Plasmid DNAs and siRNAs were transfected using Lipofectamine 2000 and Lipofectamine RNAiMAX, respectively (Life Technologies). Regarding stable transfection with a plasmid, cells were selected with 800 μg/mL G418 (Nacalai Tesque, Kyoto, Japan). The treatment of cells with D4476 was performed at a concentration of 100 μM for 3 h (Abcam, Cambridge, UK).