Ex neg m68

The HeLa S3 cell line, WI-38 cell line, and BC cell lines (5637, RT4, T24, and TCC-SUP) were obtained from the Korean Cell Line Bank (Seoul, South Korea) and American Type Culture Collection (ATCC, Manassas, VA). HeLa S3, WI-38, and 5637 cells were cultured in RPMI 1640 (Lonza, Walkersville, MD) supplemented with 10% fetal bovine serum (FBS, Gibco BRL, Grand Island, NY) and 1% penicillin/streptomycin (HyClone, GE Healthcare, Little Chalfont, UK). RT4 and T24 cells were cultured in Dulbecco's Modified Eagle's Medium (Welgene, Daegu, South Korea), and TCC-SUP cells were cultured in Eagle's Minimal Essential Medium (ATCC). The cells were maintained in a humidified atmosphere of 5% CO₂ at 37°C. Accutarget predesigned siRNAs specific for human CAD and scramble siRNAs purchased from Bioneer (Daejeon, South Korea) were used for knockdown of CAD expression. A plasmid expressing CAD transcript variant 2 (#EX-Z0014-M68, designated as ‘pCADv2’) and the blank vector (#EX-NEG-M68) were purchased from GeneCopoeia (Rockville, MD). Transfections were performed using Lipofectamine 3000 or Lipofectamine RNAiMAX transfection reagent, according to the manufacturer's instructions (Invitrogen, Carlsbad, CA).

The HCT116, HT29, CCD-18Co, and SKOV3 cells were purchased from the Korean Cell Line Bank (Seoul, Republic of Korea). Cell culture and cell line authentication were performed as previously described [9 (link)]. ICG-001 was synthesized by Wuxi AppTec (Shanghai, China). The MEIS1 vector (EX-P0088-M68) and control empty vector (EX-NEG-M68) were purchased from GeneCopoeia (Rockville, MD, USA). The generation of luciferase-tagged HCT116 (HCT116-luc) cells was previously described [31 (link)].

Lipofectamine 2000 reagent from Invitrogen was used according to the manufacturer’s instructions. ABCB9 (EX-T8156-M68) overexpression plasmid and vector control (EX-NEG-M68) were purchased from GeneCopoeia and stably transfected into the NCI-H2452 cell line under 2 μg/mL puromycin (Sigma Aldrich) selection for 14 days. The surviving cells were kept under 0.5 μg/mL puromycin maintenance selection while culturing.