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. 2013 Oct 3;8(10):e76247. doi: 10.1371/journal.pone.0076247

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© 2013 Feliciano et al

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) Upper panel: miRNA expression levels of miR-125b assessed by qRT-PCR in different cell lines. The graph shows data normalized to RNU24 levels. The relative expression of miR-125b normalized to RNU24 was calculated with the 2^-ΔΔCt method. HEK293T cells were assigned the value of 1 as they express the lowest relative RNA level of miR-125b in comparison with the other cells lines shown. Lower panel: miRNA expression levels of miR-125b assessed by qRT-PCR in different cell lines. miRVec-125b-overexpressing cells compared to miRVec-GFP control cells in MCF7, MDA-MB-231, and HMEC cells. These cell lines were pooled heterogeneous clones of the indicated genes after stable expression and selection with blasticidin for 12 days. (B) Cell numbers upon miRVec-125b overexpression compared with those of miRVec-GFP control cells in the indicated cell lines after stable expression and selection of the indicated genes with blasticidin for 12 days. (C) Phenotypic morphology of miRVec-GFP- and miRVec-125b-transduced MCF7 cells (right panel) and crystal violet staining of miRVec-GFP- and miRVec-125b-transduced MCF7 cells (left panel). Cells were selected as indicated in B. (D) Apoptosis rates analyzed by FACS of miRVec-GFP- and miRVec-125b-transduced MDA-MB-231 cells. Note the increases in early (1.3 ◊ 38.9%) and late (2.6 ◊ 26.3%) apoptosis in miRVec-GFP- and miR-125b-overexpressing cells. (E) The cell cycle profiles of miRVec-GFP- and miRVec-125b-transduced HMEC, MCF7, and MDA-MB-231 cells. miRVec-GFP-HMEC (G0/G1, 66.2%; S, 7.6%; G2/M, 26.2%) versus miRVec-125b-HMEC (G0/G1, 54.3%; S, 8.2%; G2/M, 37.4%); miRVec-GFP-MCF7 (G0/G1, 75.8%; S, 18.3%; G2/M, 5.8%) versus miRVec-125b-MCF7 (G0/G1, 48.9%; S, 22.8%; G2/M, 28.2%), and miRVec-GFP-MDA-MB-231 (G0/G1, 52.1%; S, 36%; G2/M, 11.9%) versus miRVec-125b-MDA-MB-231 (G0/G1, 46.9%; S, 30.5%; G2/M, 22.7%) (F) Representative images of colonies of the soft agar colony assay in miRVec-GFP- and miRVec-125b-transduced MCF7 cells. ***p < 0.001.