Cell proliferation of CD8+, CT26, Treg, 4T1, CT26, LS174T, MCF7, MDA231, and RKO cells was analyzed by bromodeoxyuridine (BrdU) enzyme-linked immunosorbent assay (ELISA) (Roche) according to the manufacturer’s instructions. Cells (5000 per well) were seeded in 50 μl of Opti-MEM medium (Invitrogen), while 0.5 μl of each compound at 200× was diluted in 50 μl of 2% FBS Opti-MEM medium. BrdU labeling solution (10 μl per well) was added if the cells were cultured in 100 μl per well (final concentration, 10 μM BrdU), and cells were reincubated for an additional 2 to 24 hours at 37°C. Sample absorbance was measured using an ELISA reader at 450 nm (reference wavelength of 690 nm). Each experiment was repeated three times.

Cell viability of Colo320DM and CT26 was determined (CellTiter-Glo, Promega) according to the manufacturer’s protocol. Cells (5000 per well) were seeded in 50 μl of Opti-MEM medium (Invitrogen), while 0.5 μl of each compound at 200× was diluted in 50 μl of 2% FBS Opti-MEM medium. The assay plate was incubated for 72 hours at 5% CO2 and 37°C. CellTiterGlo reagent (100 μl) was added, and 100 μl of this final mixture was analyzed for luminescence according to the manufacturer’s recommendations. Assay readout was determined by Enspire (PerkinElmer). Each experiment was repeated three times.

Testing compounds included hsBCL9CT-24 [trifluoroacetic acid (TFA) salts], LGK-974 (Selleck), ICG-001 (Selleck), erlotinib (LC Laboratories), 5-FU (ApexBio), and oxaliplatin (ApexBio). GraphPad Prism was used for data analysis.

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