Myoferlin in Breast Cancer EMT & Invasion Figure 1. Myoferlin expression in breast cancer. (A) Quantitative RT-PCR results of MYOF mRNA levels in a panel of breast cancer cell lines, normalized to 18 S and compared to the nonmalignant breast epithelial cell line MCF-10A (mean 6 s.d., n = 3 per cell type). No statistical significance was detected in comparing pairs of cell lines (Kruskal-Wallis with Dunn’s multiple comparison test). (B) Representation of the data in subpanel A grouped by known invasive capacity of the cells (2 tailed, p = 0.003, Mann Whitney). (C) Immunoblotting for MYOF in breast cancer cell lines. Densitometry measurements was done by normalizing the density of MYOF to respective GAPDH or actin staining, and further normalized to the nonmalignant mammary epithelial cell line MCF-10A (n = 4, no statistical significance detected by Kruskal-Wallis with Dunn’s multiple comparison test). An immunoblot image is shown in the subpanel. (D) Myoferlin knockdown in breast cancer cells. Immunoblot showing the expression of MYOF in MDA-MB-231 wild type (WT), lentiviral transduction control (LTV-ctrl), and MYOF knockdown cell lysates using three Sigma-Aldrich MISSIONH shRNA constructs (#1522, 10628, and 10630). doi:10.1371/journal.pone.0039766.g001 formed more cell clusters relative to control cells. When examined in greater detail using atomic force and scanning electron microscopy, the morphological divergence between 231MYOF-KD and control cells was very prominent (Figure 3). At the cellular level, 231LTV-ctrl cells often appeared elongated with tapering cytoplasmic poles, having one or more small lamellipodia and relatively short filopodia, and actin filaments oriented along the long axis of the cell (Figure 3). In contrast, 231MYOF-KD cells exhibited a flattened, polygonal shape with broad lamellipodia, and elongated filopodia that tended to orient toward neighboring cells. Collectively, these results suggested a potential reversion of epithelial-mesenchymal transition, i.e., mesenchymal-epithelial transition (MET), at the morphology level. The MET-like morphological changes prompted us to examine whether the expression of common epithelial-mesenchymal transition (EMT) markers (e.g. vimentin, fibronection, N-cadherin, and E-cadherin) was affected by MYOF depletion. By immuno- blotting, 231MYOF-KD cells expressed higher levels of the epithelial marked E-cadherin, and lower levels of mesenchymal markers, fibronectin and vimentin, than 231LTV-ctrl cells (Figure 4). MDA- MB-231 cells do not natively express N-cadherin, so it was not surprising that N-cadherin was undetectable in 231LTV-ctrl and 231MYOF-KD cells (data not shown). The results of this screen of EMT markers suggest that the morphologic shift associated with MYOF-depletion may be correlated with MET changes at the molecular level. In a survey using a qRT-PCR-based array of PLoS ONE | www.plosone.org 3 June 2012 | Volume 7 | Issue 6 | e39766