Triphala causes DNA damage and activates p53 in Capan-2 cells. A) Cells were treated with Triphala for 24 h and 60 μg total protein was separated by SDS-PAGE. The expression of p-H2A.X (Ser-139), p-ATM (Ser-1981), p-p53 (Ser-15), p53, and p21 were detected using appropriate antibodies. The blot was stripped and reprobed with the anti-actin antibody to ensure the equal protein loading. B) Time-dependent treatment cells with 60 μg/ml Triphala. C) Transcriptional activity of p53 in cells treated with Triphala for 24 h using TransAM p53 transcription assay kit. D) Effect of p53 inhibitor on Triphala induced apoptosis. Cells were pretreated with 30 μM pifithrin-α, p53 specific inhibitor for 1 hour followed by exposure to 60 μg/ml Triphala for 4 h in the presence of inhibitor. Apoptosis was determined by western blot or cell death detection kit. These assays were performed 2–3 times independently and obtained similar results. Statistical significance was determined by one-way ANOVA followed by Bonferroni's post hoc analysis for multiple comparisons. * Significantly different compared with PBS-treated control (P < 0.05). **Significantly different compared with Triphala treatment (P < 0.05).