per immunoblotting procedures as detailed in the previous section, using anti-human mouse monoclonal antibodies against MMP-1 (MAB901; R&D Systems) and fibronectin (610077; BD Trans- duction Laboratories, San Jose, CA). MMP Expression Array and MMP1 ELISA ECM proteins (MMP1, -2, -3, -8, -9, -10, and -13 and TIMP1, - 2, -3, and -4) in conditioned, serum starved supernatants of lentiviral-transduction control (MDA-MB-231LTV-ctrl) and MDA- MB-231MYOF-KD cells were determined by an antibody array (RayBioH human matrix metalloproteinase antibody array 1, RayBiotech, Inc., Norcross, GA) following the manufacturer’s protocol. Each array membranes contained duplicates for each ECM protein along with positive and negative controls. The blots were imaged and quantified by densitometry (Quantity One software, Bio-Rad), and values normalized to total protein of the corresponding cell lysates. Secreted pro-MMP-1 in cultured media of cells was detected by quantitative ELISA (DMP100; R&D Systems). Conditioned cultured media were collected by plating equal densities of each cell type in 100 mm culture dishes followed by serum-starvation for 24 h. Unconcentrated, conditioned media samples were run in duplicate in the ELISA and processed according to manufacturer’s instructions. Colorimetric results were read at 450 nm with a wavelength correction of 570 nm in a MRX Microplate Reader (Dynex Technologies, Chantilly, VA). MMP-1 concentrations were determined by interpolation using human recombinant pro- MMP-1 as a standard. In cases where the secreted pro-MMP1 was undetectable, it was assigned the assay’s lowest limit of detection (0.021 ng/ml). MTS Proliferation Assay and Cell Cycle Analysis Cells were plated in 96-well plates at a density of 2500 and/or 5000 cells per well and assayed at 24, 48, and 72 h after plating using the CellTiter 96 AQueous Non-Radioactive Cell Prolifer- ation Assay (Promega), according to the manufacturer’s instruc- tions. For flow cytometric analysis of MYOF depleted breast cancer cells, cells were fixed in 70% ethanol and stained in PBS containing 0.1% Triton X-100, 200 mg/mL of RNase, and 50 mg/mL propidium iodide (Sigma-Aldrich). DNA content was measured on a FACS Calibur or LSR II flow cytometer (Becton-Dickinson)), and data were acquired and analyzed using flow cytometer software (BD). Each analyzed sample contained at least 16106 cells. Migration and Invasion Assays Cells were washed with PBS, trypsinized, pelleted and resuspended in serum-free DMEM. Cells were then seeded onto Boyden Chamber inserts (8 mm pores; Millipore) at a density of 7.56104 cells in 24-well plates with 10% FBS containing DMEM in the bottom chamber as a chemo-attractant. In parallel, input controls were seeded with the same number of cells in the same volumes of serum-free and 10% FBS media without inserts. Cells were incubated (37uC, 5% CO2) for 24 hours. The inserts were processed by rinsing in PBS and fixation with 3.7% formaldehyde containing 0.05% crystal violet for 10 min. After repeated washes with PBS and distilled water the chambers were air-dried. Migrated cells on the bottom of the inserts were collected with cotton swabs and placed into Eppendorf tubes. Crystal violet dye was extracted from the Q-tips and input control wells with 80% methanol for 30 min, and quantified at 570 nm. Percentage migration was calculated using the ratio of the migrated cells over the total cells (input control) to determine the percentage of cell migration. For invasion, the Boyden Chamber assay was Myoferlin in Breast Cancer EMT & Invasion conducted in the same manner but with the addition of 20 ml of growth factor reduced Matrigel (BD) or 3 mg/ml collagen I (rat tail, Gibco) to the top of the insert and allowed to gel for 1 h at 37uC, 5% CO2. Quantitative Reverse Transcription PCR Array RT2 ProfilerTM PCR array (SABiosciences, Frederick, MD) was performed for EMT-associated genes (cat #PAHS-090) and human extracellular matrix and adhesion molecules (cat # PAHS-013) following manufacturer’s protocol. Briefly, total RNA was extracted as described in the qRT-PCR section from lentiviral control and myoferlin-deficient MDA-MB-231 cells that had been in culture for 10 consecutive passages. Complementary DNA was generated from 1 mg of total RNA per cell type using random hexamers and oligo-dT18 primers as part of the RT2 First Strand Kit (SABiosciences). Equal amounts of dilute cDNA was mixed with LightCyclerH 480 SYBR Green I Master mix (Roche) and aliquoted to each well of the PCR array plate containing pre- filled gene-specific primer sets, and PCR was performed according to manufacturer’s instructions for the Roche LightCyclerH 480. The LightCyclerH 480 Software (Roche Applied Science) was used to calculate the threshold cycle (crossing point, Cp) values for all the transcripts in the array. The Cp values were exported into a spreadsheet-based PCR array data analysis template (SABios- ciences) to calculate fold changes in gene expression using the DDCt method. Statistical Analysis Graphical representation and statistical analysis of data were done using Prism version 5 (GraphPad). Error bars represent standard deviation in all cases, unless differently noted. Statistical significance was determined by appropriate statistical tests following normality testing of the data, using the KS, D’Agostino & Pearson omnibus and/or Shapiro-Wilk normality tests. Com- parisons between 2 groups with Gaussian distribution were carried out with the 2-tailed student t-test for unpaired samples, while those among 3 or more groups were carried out with one-way ANOVA followed by Tukey’s Multiple Comparison Test. Data exhibiting a non-Gaussian distribution were analyzed with either the Mann Whitney statistical test (2 groups) or the Kruskal-Wallis with Dunn’s multiple comparison post-test (3 or more groups). For analysis, results were regarded as significant if p values were less than 0.05. Supporting Information Figure S1 Analysis of the myoferlin gene expression in breast cancer cells. Graphical representation (mean 6 s.d.) of expression data from two microarray probe sets for myoferlin reported in the study by Neve and colleagues [34], showing a higher expression (Kruskal-Wallis test/Dunn’s multiple compari- son analysis) in the Basal B cells compared with the luminal cells. (TIF) Figure S2 Graphical representation from ArrayExpress gene expression atlas (accession # E-TABM-276) of MYOF mRNA expression level in breast tissue samples from healthy patients and patients with invasive ductal carcinoma. (TIF) Figure S3 Myoferlin lentiviral constructs. Target and sequence information of lentiviral constructs used to generate myoferlin-deficient cell lines. Bold letters indicate coding sequence. (TIF) PLoS ONE | www.plosone.org 10 June 2012 | Volume 7 | Issue 6 | e39766