FIGURE 3.

p27^Kip1, a target of miR-221/222, imparts tamoxifen sensitivity to MCF-7 cells. A, whole cell extracts from MCF-7 cells, OHT^R cells, and miR-221/222-transfected and vector (Vec)-transfected MCF-7 cells were subjected to SDS-PAGE and probed with anti-p27^Kip1 antibody. The membrane was reprobed with anti-Ku-70 antibody, and p27^Kip1 expression was normalized to Ku-70 protein. O/E, overexpressing. B, total RNAs from MCF-7 cells, OHT^R cells, and miR-221/222-transfected and vector-transfected MCF-7 cells were analyzed by real-time RT-PCR with primers specific for p27^Kip1 and 18 S rRNA. C, whole cell extract from the p27^Kip1-transfected OHT^R cell pool was subjected to Western blot analysis with anti-p27^Kip1 antibody and reprobed with anti-Ku-70 antibody. D, OHT^R cells transfected with empty vector and p27 expression vector were treated with 5 μm tamoxifen for 72 h. Cell metabolic activity was measured every 24 h using the MTT assay. The metabolic activity of cells at 0 h was taken as 1. The results are the means ± S.D. of triplicate assays. E, vector-overexpressing (panels A–C) and p27^Kip1-overexpressing (D–F) OHT^R cells were treated with 0 and 15 μm tamoxifen for 16 h. The cells were photographed using a phase-contrast microscope. The small arrows in panels G and H indicate autophagosome-like bodies. F, whole cell extracts from the vector-transfected and p27^Kip1-transfected OHT^R cells were subjected to SDS-PAGE and probed with antibodies against caspase-7 and PARP. The blot was reprobed with anti-Ku-70 antibody to normalize protein loading. The signal in each band was quantified using Kodak Imaging software. Quantification of uncleaved and cleaved caspase-7 and PARP is presented in the bar diagrams. The results are representative of three independent experiments. TAM, tamoxifen.