Figure 6. Myoferlin depletion attenuates MMP1 expression and collagen I invasion capacity of MDA-MB-231 cells. (A) Representative (n = 3) immunoblotting results of secreted MMP1 in 24 h serum starved supernatant of MDA-MB-231 lentiviral control (LTV-ctrl) and myoferlin knockdown (MYOF-KD) cells. Recombinant human matrix metalloproteinase-1 was used as a standard. Verification of myoferlin knockdown was done in the corresponding cell lysates with GAPDH as a loading control. (B) Secreted pro-MMP1 was evaluated in 231LTV-ctrl and 231MYOF-KD cells (n = 2) using ELISA (mean 6 s.d., Kruskal-Wallis test/ Dunn’s multiple comparison analysis). (C) Results from Boyden chamber invasion assays using a coating of 3 mg/ml of rat tail collagen I to Myoferlin in Breast Cancer EMT & Invasion evaluate the invasive capacity of 231LTV-ctrl and 231MYOF-KD cells (n = 3, mean 6 s.d., Kruskal-Wallis test/Dunn’s multiple comparison analysis). doi:10.1371/journal.pone.0039766.g006 public domain and verified that none of the shRNAMYOF constructs predicted the targeting of any nucleic acid sequences within the human MMP-1 gene. In addition, we found MMP1 expression to be also down-regulated when MYOF expression was knocked down using an alternative shRNA construct with similar knock down efficiency. Another unanticipated finding in the current studies was the partial reversion of the mesenchymal-like shape in wild-type and control MDA-MB-231 cells to a more epithelial-like phenotype in 231MYOF-KD cells. We verified this potential MET event using immunofluorescence, scanning, and atomic force microscopy. In addition, we also demonstrated the likelihood of an MET in the setting of depleted MYOF using immunoblotting that revealed down-regulation of fibronectin and vimentin and up-regulation of E-cadherin. In a recent paper from our group, MYOF depletion was shown to lead to the down-regulation of the phosphorylation of several receptor tyrosine kinases, including EphB4, FGFR2, Hck, IGF-IR, JAK2, TXK, VEGFR2 [30]. This observation is in agreement with the work of Demonbreun et al. demonstrating that loss of MYOF led to impaired insulin-like growth factor I receptor (IGF- IR) signaling in a murine model [51]. In separate reports, MYOF was shown to be required for Tie-2 (angiopoietin-1 receptor) activation [25] and vascular endothelial growth factor receptor 2 (VEGFR-2) expression in vascular endothelial cells [24]. More- over, Sharma et al., showed that disruption of dysferlin, a close molecular cousin of MYOF, in endothelial cells led to poly- ubiquitination and proteasomal degradation of platelet endothelial cellular adhesion molecule-1 (PECAM-1/CD31) signaling [52]. These data, when considered collectively and in the context of our breast tumor cell investigations, implicate MYOF as a critical participant in key processes within the intracellular vesicle trafficking mechanism including cell motility, MMP expression/ secretion, and RTK activation. Our work is the first examination of the role of MYOF in cancer cell biology and may shed light on essential features of vesicle shuttling of important cargos within tumor cells that participate in cancer progression and metastasis. Materials and Methods Cell Culture Methods and Cell Line Authentication Human MCF-10A (generously provided by Dr. Joan Brugge, Boston, MA, [32,53]), MCF-7 (HTB-22, ATCC, Bethesda, MD), MDA-MB-231 (HTB-26, ATCC), T47D (HTB-133, ATCC), and BT549 (generous gift from Dr. Lisa Yee, Columbus, OH; HTB- 122, ATCC) cells were used in this study. MCF-10A cells were maintained in Dulbecco’s Modified Eagle Medium Ham’s F12 (DMEM/F12, Gibco, Carlsbad, CA) supplemented with 5% horse serum (Gibco), 20 ng/ml hEGF (PeproTech, Rocky Hill, NJ), 0.5 mg/ml hydrocortisone (Sigma-Aldrich, St. Louis, MO), 100 ng/ml cholera toxin (Sigma-Aldrich), 10 mg/ml bovine insulin (Sigma-Aldrich), and 1% penicillin/streptomycin (Gibco). MCF-7 and MDA-MB-231 cells were maintained in DMEM with 4.5 g/L D-glucose supplemented with 10% fetal bovine serum (FBS, Gibco). T47D and BT549 cells were cultured in RPMI-1640 with 10% FBS. The cell lines used in this study were authenticated by DNA profiling using short tandem repeat (STR) analysis on a PowerPlex 1.2 System (Promega, Madison, WI) at John Hopkins University (Fragment Analysis Facility, Baltimore, MD). Fur- PLoS ONE | www.plosone.org 8 June 2012 | Volume 7 | Issue 6 | e39766