Figure 3

Treatment with SXR activators increases expression of p53 and p53 target genes. Total RNA was isolated from A) MCF-7 cells or B) ZR75-1 cells grown in the presence of 10 μM SXR activators rifampicin, anandamide, or clotrimazole (RIF, ANA or CLO) for 72 hours and 24 hours respectively. RNA was reverse transcribed and analyzed by QRT-PCR using primers for human p53, p21, BAX and PUMA. Data are depicted as average fold induction relative to solvent control in triplicates ± S.E.M. These results were replicated in at least three independent experiments. *represents P ≤ .05, **represents P ≤ .01 and # represents P ≤ .001 (by one-way ANOVA analysis). C) SXR activation causes p53 accumulation. Cell lysates made from C) MCF-7 and D) ZR-75-1 cells treated with SXR agonists (10 μM) or solvent controls for 72 and 24 hours respectively were subjected to Western blot analysis using p53 antibody (FL-393 HRP, Santa Cruz Inc.). Equal loading was confirmed by stripping and re-probing the same blot with an anti-GAPDH antibody. Camptothecin (CAMP) 24 hour treatment was used as a positive control for p53 induction. The chemiluminescent bands were acquired by Alpha Innotech Fluorchem SP imager (Alpha Innotech Inc., CA, USA) and analyzed by spot densitometry analysis using FluorChem AlphaEase FC software (Alpha Innotech). The experiment was done in duplicates and the numbers represent the average from two independent runs. Note that the gap in ANA lane of ZR-75-1 is because of gel tearing at the time of transferring.