thermore, to rule out cell line contamination as the cause for the morphology changes seen in MDA-MB-231 myoferlin- deficient cells (231MYOF-KD), the identity of the 231MYOF-KD cells as MDA-MB-231s was also authenticated through short tandem repeat profiling. Lentiviral shRNA Transduction Recombinant lentiviral particles containing non-target control shRNA and human myoferlin targeted shRNA (TRCN0000010628, TRCN0000010630, TRCN0000001522, and TRCN0000320398, Figure S3) in the pLKO.1 vector were purchased from Sigma-Aldrich (MISSIONH). The myoferlin constructs are referred to as #10628, #10630, #1522 and #320398, respectively, in the body of the manuscript. For lentiviral transduction, cells were seeded in 24-well culture plates and incubated overnight at 37uC in 5% CO2 in a humidified atmosphere. Media was replaced with media containing 8 mg/ml hexadimethrine bromide (Sigma-Aldrich) and lentiviral particles were added to subconfluent at a multiplicity of infection (MOI) of at least 1. After overnight incubation (37uC, 5% CO2), virus- containing supernatant was replaced with complete media and incubated overnight. Transduced cells were selected in media containing an appropriate puromycin concentration as predeter- mined by a puromycin kill curve. RNA Extraction and Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) Total RNA was extracted using TRIzolH reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol up to the chloroform extraction and centrifugation step. The resulting aqueous phase was mixed with an equal volume of 70% ethanol and applied to an RNeasy mini column (QIAGEN, Valencia, CA) and processed according to the manufacturer’s protocol with on column DNAse digestion. Total RNA was quantified by UV absorbance at 260 nm and 280 nm on a NanoDrop 2000 (Thermo Scientific, Waltham, MA), and 1–2 mg total RNA from each sample were reverse-transcribed to cDNA using oligo-dT18 and random hexamer primers with a Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science, Indianapolis, IN). Quantitative RT-PCR was performed using an equal amount of cDNA per sample on a LightCyclerH 480 II System (Roche Applied Science) using primer-probe sets specific to MYOF, MMP1, RLPO and 18 S rRNA (Applied Biosystems, Carlsbad, CA) with the LightCyclerH 480 Probes Master Mix, and results analyzed with the LightCyclerH 480 Software. Immunofluorescence MDA-MB-231 cells cultured on glass coverslips and serum- starved for 24 h were fixed for 1 h in 4% (w/v) paraformalde- hyde/PBS, permeabilized for 15 min with 0.2% (v/v) Triton X– 100/PBS, and blocked for 1 h in 5% normal goat serum/1% nonfat dry milk/PBS. Samples were incubated with antibodies directed against myoferlin (1:400, Sigma) overnight at 4uC. After washing, cells were exposed to fluorochrome-conjugated second- ary antibodies (Molecular Probes). After further stringent washing in PBS, actin cytoskeleton was stained with AlexaFluor 488- phalloidin (Molecular Probes) for 20 min. Nuclei were stained and cells mounted with ProLongH Gold Antifade Reagent with DAPI (Invitrogen) and visualized with confocal laser scanning microsco- py (Olympus FV 1000 Spectral Confocal system; Olympus America Inc., Center Valley, PA). Myoferlin in Breast Cancer EMT & Invasion Atomic Force (AFM) and Scanning Electron Microscopy (SEM) MDA-MB-231 lentiviral control and myoferlin-deficient cells were seeded in 60 mm cell culture dishes (5–86105 cells), and allowed to grow to 80–90% confluence for AFM analysis. Prior to AFM analysis, culture media was exchanged for CO2 independent media (Invitrogen) supplemented with 1% FBS, 1% Penicillin/ Streptomycin and 4 mM L-Glutamine, for optimal cell survival during the atomic force measurements. A Bioscope II instrument (Veeco, Plainview, NY) mounted on the stage of an Axiovert 200 inverted optical microscope (Zeiss, Oberkochen, Germany) was used in contact mode for live cell imaging. Silicon nitride (SiN) triangular cantilevers (Veeco) 200 mM in length with a tip angel h = 35u and a nominal spring constant k of 0.01 N/m were used to image the cells. Image acquisition was done at a scanning rate of 0.5 Hz with 5126512 lines resolution for recording the height and the deflection channels. All images were recorded with a scan size area of 70–80 mm2 covering ,5 cells, with 4–5 areas completed per cell type. The experiments were repeated three times and the data were analyzed using version 7.30 of the NanoScope software (Veeco). For SEM, cells cultured on thermoplastic coverslips (Therma- noxTM, Nunc, Waltham, MA) were fixed with 3% glutaraldehyde, post-fixed with 1% OsO4 and dehydrated through an ethanol and hexamethyldisilazane series. Samples were mounted onto SEM studs and prepared for SEM using a Pelco Model 3 sputter coater with gold-palladium (0.07 mbar, 17 mA, 110 s) and imaged with an FEI Nova NanoSEM microscope (FEI Company, Hillsboro, OR). Protein Extraction and Immunoblotting Cells were rinsed with PBS and lysed for 30 min in cold RIPA lysis buffer supplemented with protease inhibitor cocktail (Sigma- Aldrich) and 1 mg/ml of pepstatin A (USB, Santa Clara, CA). Cell lysates were centrifuged for 10 min at 17,0006g and 4uC. Supernatant aliquots were assayed for protein concentration using the BCA Protein Assay (Pierce, Rockford, IL) with bovine serum albumin as a standard. Total protein from each lysate (30 mg per lane) were resolved by SDS-PAGE and transferred to nitrocellu- lose membranes. Non-specific binding was blocked by incubation in Tris-buffered saline (pH 8) containing 0.1% Tween-20 and 5% non-fat dry milk. Membranes were probed with primary antibodies against: b-actin (Santa Cruz Biotechnology, Santa Cruz, CA), GAPDH (Chemicon, Billerica, MA), myoferlin (Sigma- Aldrich), E-cadherin (EP700Y, Abcam, Cambridge, MA), vimen- tin (Sigma-Aldrich V6630), or MMP1 (R&D Systems, Minneap- olis, MN) diluted in blocking solution. After washing, membranes were exposed to horseradish peroxidase-conjugated secondary antibodies and immune complexes were revealed with Super- Signal West Femto chemiluminescent substrate (Pierce), visualized using the VersaDoc Imaging System and analyzed with Quantity One analysis software (Bio-Rad, Hercules, CA). Supernatant Collection and Immunoblotting Cell conditioned media were collected after the cells were plated at equal densities in 100 mm dishes, then serum starved in 4 ml of basal media for 24 h. The conditioned media were concentrated 50-fold with Amicon Ultra-4 10-kDa centrifugal filters (Millipore, Billerica, MA), and equal volumes of concentrated media were reconstituted in sample buffer and resolved by SDS-PAGE. Human recombinant MMP1 (rhMMP-1) from conditioned media of rhMMP-1 expressing NS0 mouse myeloma cells (WBC024; R&D Systems) was used as a positive control. Gels were processed PLoS ONE | www.plosone.org 9 June 2012 | Volume 7 | Issue 6 | e39766