5 fluorouracil

The chemosensitivity assay was performed as previously described.^{28 (link)} Cells were seeded at a density of 4 × 10³ cells/well in 96-well plates, and precultured for 24 h. Next, the cells were exposed to various concentrations of 5-fluorouracil (5-FU; Tokyo Chemical Industry Co., Ltd, Tokyo, Japan) and oxaliplatin (L-OHP; Yakult Honsha Co., Ltd, Tokyo, Japan) for 72 h at 37 °C in a humidified incubator containing 5% CO₂. We evaluated the in vitro cytotoxic effects of 5-FU and L-OHP using the Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan), following the manufacturer’s protocol. The viability data of CRC cells treated with each concentration of L-OHP were used to calculate the half-maximal inhibitory concentration values.

AR-A01441817 (link), the GSK-3β inhibitor XXVI, and a cdk4 inhibitor were from Merck (Darmstadt, Germany). Docetaxel and 5-fluorouracil were from Tokyo Chemical Industries (Tokyo, Japan). KU0063794, LY294002, lithium chloride, and other chemicals not otherwise specified were from Wako Pure Chemical Industries (Tokyo, Japan).

Reagents were obtained as follows: Navitoclax and A1210477 from MedChemExpress; venetoclax and S63845 from Chemietek; topotecan from Toronto Research Chemicals; olaparib, etoposide, and vincristine from Selleck Chemicals; 5-fluorouracil from Tokyo Chemical Industry; and CHAPS from Millipore-Sigma. Antibodies were purchased from the following suppliers: anti-PUMA (#sc-374223, 1:1000) and anti-actin (goat polyclonal, I-19, #sc-1615, 1:500) antibodies from Santa Cruz Biotechnology; anti-NOXA antibody from ENZO Life Sciences (#ALX-804-408-c100, 1:1000); anti-BCL2 antibody from DAKO (#M0887, 1:1000); anti-BAK antibody from Millipore (#06-536, 1:1000) and antibodies to BAX (#2772S, 1:1000), BCLX_L (#2764S, 1:1000), BIM (#2933S, 1:1000), MCL1 (#4572S, 1:1000), caspase-3 (#9662, 1:1000), caspase-9 (#9502, 1:1000), and tubulin (#2148, 1:1000) from Cell Signaling Technology. Rat anti-BID antibody was a kind gift from David Huang (Walter & Eliza Hall Institute, Melbourne, Australia).