Figure 4. AM increases p53 expression when added before but not after irradiation by delaying p53 degradation but not inhibiting p53 transcription.

IEC-6 cells were treated with AM (4 mM) or vehicle (PBS) for 60 minutes, after which these compounds were washed out. Cells were irradiated using 18 Gy. Protein and mRNA levels of p53 were analyzed using (A) Western blotting and (B) RT-PCR, respectively, at different time points. The assay for p53 degradation in (C) p53 wild type cells (IEC-6 cells and HCT 116 cells) and (D) p53 mutant cells (DLD-1 cells and COLO 205 cells) was performed 60 minutes after AM (4 mM) or vehicle (PBS) administration, after which the AM and vehicle were washed out. After treatment with 50 μg/mL cycloheximide (CHX) for different durations, the cells were harvested, and Western blotting for p53 expression was performed.