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. 2014 Apr;34(7):1246–1261. doi: 10.1128/MCB.01216-13

FIG 4.

FIG 4

SMRT coactivates p53 transcriptional activity in MCF-7 cells. Cells were transfected with 20 pmol of control siRNA or SMRT-specific siRNA (siSMRT), prior to transfection with 1 μg of either the p21 (A) and MDM2 (B) luciferase reporter genes, and treated with vehicle (Veh; 0.1% ethanol) or 1 nM E2 for 24 h. Luciferase activity data are the means ± SEMs of three (p21) and two (MDM2) experiments. (C) MCF-7 cells were transfected with 500 ng of a Flag-tagged expression vector for SMRT or an empty-vector control (basal) along with 500 ng of the p21-Luc reporter gene. Cells were harvested 48 h after transfection, and luciferase activity was measured. The activity for SMRT is shown relative to that for the empty-vector control. Values represent the averages ± SEMs (n = 4). (D) MCF-7 cells were transfected with control siRNA or SMRT-specific siRNA, and 48 h later, the cells were treated with vehicle (PBS; without doxorubicin [−Dox]) or doxorubicin (+Dox; 1 μM) for 16 h. Whole-cell lysates were resolved in SDS-polyacrylamide gels, and the expression of the p21, p53, SMRT, and actin proteins was analyzed by Western blotting. (E) ZR75-1 cells were transfected with 30 pmol of control siRNA or SMRT-specific siRNA. After 48 h, the cells were treated with vehicle (PBS) or doxorubicin (1 μM) for 16 h, followed by RNA isolation and quantitation by RT-qPCR. The p21 mRNA levels were normalized to the signals obtained for 18S RNA. The changes in the mRNA levels are represented relative to the level of control siRNA in doxorubicin-treated cells. Values represent the means ± SEMs of data from two independent experiments. (F) Saos-2 cells were transfected with control siRNA (siC) or SMRT-specific siRNA (siS), and 48 h later, the cells were treated with vehicle (PBS without doxorubicin) or doxorubicin (+Dox; 0.5 μM) for 16 h. Whole-cell lysates were resolved by SDS-PAGE, and the expression of p21, BRCA1, SMRT, and actin proteins was analyzed by Western blotting. (G) MCF-7 cells were transfected with 500 ng of either the 14-3-3σ wild-type (WT) or mutant (MT) luciferase reporter genes, along with 500 ng pCR3.1 (basal) or full-length human SMRT plasmids. After 24 h, cells were treated with vehicle or 1 μM doxorubicin for 24 h. Luciferase activities are presented relative to those obtained for cells transfected with wild-type 14-3-3σ–Luc and SMRT and treated with doxorubicin. Values represent the averages ± SEMs (n = 3). RLU, relative light units.