FIG 8.

Effect of HDAC3, GPS2, and TBL1 depletion on p21 mRNA levels. MCF-7 cells were transfected with 20 pmol of control siRNA or siRNA specific for GPS2 (A), TBL1 (B), or HDAC3 (C). After 48 h, the cells were treated with vehicle (PBS; filled bars) or doxorubicin (0.5 μM; white bars), and p21 mRNA levels were quantitated by RT-qPCR and normalized to the signals obtained for 18S RNA. The changes in the p21 mRNA levels were represented relative to the level of control siRNA in doxorubicin-treated cells. Values represent the means ± SEMs of data from four (GPS2 and HDAC3) and three (TBL1) independent experiments. (D) Effect of HDAC3 depletion on p53 levels. MCF-7 cells were transfected with 20 pmol of control or HDAC-specific siRNA, followed by treatment with vehicle or doxorubicin for 24 h. Cells were lysed, and proteins were resolved in SDS-polyacrylamide gels and immunoblotted with p53 and actin (loading control) antibodies. (E) Chromatin immunoprecipitation assay showing doxorubicin-induced recruitment of p53 at the p21 gene promoter relative to that at enhancer sites. MCF-7 cells were treated with vehicle (PBS) or doxorubicin (1 μM) for 6 h, followed by chromatin isolation and immunoprecipitation using p53 antibody in parallel with the IgG negative control. Immunoprecipitated DNA was quantitated by qPCR using the indicated primers. Data represent averages ± SEMs of three independent experiments. The changes in the recruitment levels were represented relative to the amount of p53 recruited to the 5′ enhancer site following doxorubicin treatment. (F) Effect of NCoR depletion in MCF-7 cells on p21 mRNA levels assessed by RT-qPCR. Cells were transfected with control siRNA or NCoR siRNA, and p21 mRNA levels were quantitated as described for panels A to C. P values were determined by Student's t test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.0001.