Figure 4.

TruMBD4 enhances 5FU cytotoxicity in hMLH1-proficient cells. (A) Establishment of stable, TruMBD4-expressed HT29 cell clones as shown by Western blot (right lane). Cells were transfected with a pcDNA3 plasmid (Invitrogen) encoding TruMBD4, and selected by G418. HCT116 lysates served as a positive control since it expresses both normal MBD4 protein and TruMBD4 (left lane). HT29 cells transfected with an empty pcDNA3 plasmid served as negative control (middle lane). β-actin served as a loading control. (B) MTS assay. Cells were seeded at a density of 5000 cells per well into 96-well plates in culture medium treated with 5 μM, 10 μM;, 15 μM;, 20 μM of 5FU. After 5 d of growth, the number of viable cells was counted via the assay. (C) Clonogenic assay. Cells were plated in growth medium supplemented by 10% FBS and containing various concentrations of 5-FU (0, 5, and 10 µM). After 10 d of growth, the culture plates were washed, fixed with methanol, and stained with 3% Giemsa. From both MTS and clonogenic assays, TruMBD4 enhances 5FU-induced cytotoxicity.