Stable knockdown of miR-200a facilitates CSC-like phenotypes in WB cells.

<p>(A) Growth curve of WB-miR-NC and WB-anti-miR-200a cells determined by cell counting. Data are expressed as the mean ± SD; n = 3; *, p<0.05. (B) Representative images of spheroids formed by WB-miR-NC and WB-anti-miR-200a cells in the spheroid formation assay (left, magnification×100). Number of spheroids formed in the primary, secondary and tertiary generations of suspension cultured WB-miR-NC or WB-anti-miR-200a cells (right). Data represent means from four randomly selected fields under the microscope, and error bars represent SD. *, p<0.05; **, p<0.01. (C and D) Apoptosis of WB-miR-NC and WB-anti-miR-200a cells measured by caspase-3/7 assay and Annexin V and PI staining. Data are expressed as the mean ± SD (C) and representative dot plots of apoptosis tests are shown (D); n = 5. (E) Expression of EpCAM, CD133, ABCG2, CK19, AFP, ALB and c-myc in WB-anti-miR-200a cells measured by qRT-PCR. Data are normalized to β-actin, shown relative to the level in WB-miR-NC cells and expressed as the mean ± SD; n = 3; *, p<0.05; **, p<0.01. (F) WB-miR-NC and WB-anti-miR-200a cells were treated with 10 ng/ml paclitaxel or 30 ng/ml doxorubicin for 48 h and then subjected to FACS with Annexin V and PI staining, respectively. Representative dot plots (left) and the mean percentage of apoptotic cells (± SD) from three independent experiments (right) are shown; *, p<0.05; **, p<0.01.