CIP2A/PP2A/p-Akt mediated tamoxifen-induced apoptosis. (A) Ectopicexpression of myc-tagged Akt protected MDA-MB-231 cells from tamoxifen-induced apoptosis. (B) Ectopic expression of myc-tagged CIP2A protected MDA-MB-231cells from tamoxifen-induced apoptosis. Note that cells with ectopic expression of myc-tagged CIP2A also had constitutively high p-Akt. For experiments (A) and (B), cells were transfected as described in Material and Methods. Control cells were empty-vector cells. Apoptosis was analyzed by flow cytometry after cells were sequentially exposed to DMSO or tamoxifen at the indicated doses for 36 hours. (C) Analysis of PP2A activity in drug-treated cells. Cells were treated with DMSO ortamoxifen at 7.5 μM or okadaic acid at 20 nM (as a negative control) or forskolin 40μM (as a positive control) for 36 hours. Cell lysates were assayed for PP2A activity. (D) Pretreatment of PP2A inhibitor okadaic acid protected cells from tamoxifen-induced apoptosis. Cells were treated with DMSO (control) or tamoxifen (7.5 μM) for 36 hours. For pretreatment, cells were pretreated with okadaic acid(20nM) for 1 hour; then they were washed and treated with tamoxifen (7.5 μM) for 36hours. Cell lysates were separated and assayed for sub-G1 analysis and westernblotting. (E) Cotreatment of tamoxifen with forskolin enhanced apoptosis in resistant HCC-1937 cells. Cells were treated with DMSO (control), tamoxifen (7.5 μM), or co-treated with tamoxifen (7.5 μM) and forskolin (40 μM) for 36 hours. Cell lysates were separated and assayed for sub-G1 analysis and western blotting. (F) Downregulation of CIP2A by siRNA increased tamoxifen-induced apoptosis in HCC-1937 cells. Cells were transfected with either control (scrambled siRNA) or CIP2A siRNA for 72 hours followed by exposure to tamoxifen at 7.5 μM for 36 hours. For (A) to (F), Columns, mean (n = 3); bars, SD; *P< 0.05.