Figure 4

Expression, MYB regulation and function of BCL2 in MCF-7 cells. (a) BCL2 expression in MCF-7 cells treated with increasing doses sodium butyrate (NaBu). After 72 hours of treatment, cells were harvested and the mRNA was analyzed for expression of BCL2 by quantitative (Q) PCR. (b) MCF-7 cells stably transduced with an inducible MYB shRNA vector were grown in the presence of doxycycline (Dox) for three days, after which BCL2 was analyzed by Q-PCR. Standard deviation is represented by error bars. (c) A schematic of the BCL2 gene (not to scale) showing the relative locations of the primers (MBS-1-4) used for ChIP assays. (d) Q-PCR of MCF-7 cells assessed by ChIP assays using anti-MYB Ab. Rabbit IgG was used as a negative control. MYC is used as a positive control, and GAPDH and an upstream primer set are used as negative controls. (e) Western blot analysis of BCL2 knockdown by four siRNAs (#1-4), parental cells, and control siRNA. MCF-7 s were transiently treated with the siRNAs and at 24 hours, assessment of their ability to reduce BCL2 was undertaken. A ß-tubulin loading control is also shown. Results are indicative of two independent experiments. (f) MCF-7 cells treated with four independent BCL2 siRNAs were grown for 24 hours with 0, 0.1, or 1 mM NaBu as indicated. TUNEL assays were analyzed by flow cytometry to quantify the number of apoptotic cells. Standard deviation is represented by error bars.