Primary antibodies to E-cadherin (clone 36) and FAK (clone 77) were purchased from BD Transduction Laboratories (Lexington, KY). Primary antibodies to N-cadherin, ezrin, moesin (Q480), pan-ERM, phospho-ERM, and phospho-MLC (Ser19; mouse monoclonal) were purchased from Cell Signaling Technology (Beverly, MA). Primary antibodies to radixin, fibronectin, α-SMA (clone 1A4), and α-tubulin (clone DM1A) were purchased from Sigma-Aldrich (St. Louis, MO). Primary antibody to CD44 (clone A020) was purchased from Calbiochem (La Jolla, CA). Primary antibody to phospho-MLC (S19/S20; rabbit polyclonal) was purchased from Rockland Immunochemicals (Gilbertsville, PA). Primary antibodies to p34-Arc/ARPC2 and β-actin (clone C4) were purchased from Millipore (Billerica, MA). Primary antibodies to FAK-pY397 and secondary antibodies conjugated to Alexa Fluor 488 or Alexa Fluor 568 were purchased from Invitrogen (Carlsbad, CA). Secondary antibodies conjugated to peroxidase were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA).
Cell culture, treatments, and transfections
NMuMG normal mouse mammary gland epithelial cells (obtained from R. Derynck, University of California, San Francisco) and A549 human lung adenocarcinoma cells (obtained from H. Chapman, University of California, San Francisco) were maintained in DME medium (4.5 g/l glucose) supplemented with 10% fetal bovine serum (FBS; Invitrogen), 100 U/ml penicillin, and 100 μg/ml streptomycin. Growth medium for NMuMG cells was also supplemented with 10 μg/ml insulin (Sigma-Aldrich). MCF-10A human mammary epithelial cells (obtained from J. Debnath, University of California, San Francisco) were maintained in DME/F-12 medium (4.5 g/l glucose) supplemented with 5% horse serum (Invitrogen), 10 μg/ml insulin, 20 ng/ml epidermal growth factor (PeproTech, Rocky Hill, NJ), 0.5 μg/ml hydrocortisone (Sigma-Aldrich), 100 ng/ml cholera toxin (Sigma-Aldrich), 100 U/ml penicillin, and 100 μg/ml streptomycin. 293TA human embryonic kidney cells were maintained in DME medium (4.5 g/l glucose) supplemented with 10% tetracycline-free FBS (Clontech, Mountain View, CA) and 110 mg/l sodium pyruvate. All cell lines were maintained at 37°C in 5% CO2.
Unless otherwise indicated, NMuMG cells were treated with 5 ng/ml recombinant human TGF-β (in 10 mM citric acid, pH 3; PeproTech) for 48 h to induce EMT. MCF-10A and A549 cells were treated with 10 ng/ml TGF-β for 3–5 d or for 1–2 d in serum-free small airway basal medium (Lonza, Basel, Switzerland), respectively. To inhibit TGF-β type I receptor signaling, cells were treated with 5 μM SB431542 (in dimethyl sulfoxide [DMSO]; Sigma-Aldrich). To inhibit Rho kinase (ROCK), cells were treated with 5 μM Y-27632 (in dH2O; Calbiochem) for 45 min before TGF-β treatment. For short-term treatments with pharmacological inhibitors, cells were incubated with 10 μM Y-27632, 5 μM blebbistatin (in DMSO; Calbiochem), or 5 μM nocodazole (in DMSO; Calbiochem) for 1 h after 48 h with TGF-β. Cells were transfected using Lipofectamine 2000 (Invitrogen), according to the manufacturer's protocol. Transfected cells were seeded on glass coverslips and cultured for 2–3 d before experimental analysis.
DNA constructs, lentivirus production, and generation of stable cell lines
The plasmid containing mEGFP-N1-LifeAct sequence was kindly provided by Roland Wedlich-Sölder (Max Planck Institute of Biochemistry, Martinsried, Germany). The moesin-GFP construct was kindly provided by Francisco Sanchez-Madrid (Universidad Autonoma de Madrid, Madrid, Spain). Lentiviral plasmids (pLKO.1-puro) containing shRNA sequences to mouse moesin were obtained from Sigma-Aldrich (NM_010833, MISSION shRNA). MISSION Non-Target shRNA Control Vector (Sigma-Aldrich) was used as a control. Lentiviruses were produced in 293TA packaging cells using the Lenti-X HT Packaging System (Clontech), according to the manufacturer's protocol. For lentiviral transduction, NMuMG cells were infected with lentivirus expressing control or moesin shRNA in growth medium supplemented with 4 μg/ml Polybrene (Sigma-Aldrich). Stable clonal cell lines were selected with 10 μg/ml puromycin (Cellgro; Mediatech, Manassas, VA) and were maintained in 2.5 μg/ml puromycin.
Immunoblot analyses were performed using lysates from cells lysed in ice-cold RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.5% NaDOC, 0.1% SDS, and protease inhibitors) containing phosphatase inhibitors (50 mM NaF, 1 mM NaVO4, 1 mM ethylene glycol tetraacetic acid, 10 mM sodium pyrophosphate, 1 mM glycerol phosphate, and 10 nM calyculin A). Protein concentrations of clarified cell lysates were determined using a bicinchoninic acid protein assay kit (Pierce, Thermo Scientific, Rockford, IL). Proteins (5–10 μg) were separated by SDS–PAGE and transferred to polyvinylidene fluoride membranes. Membranes were blocked in 5% milk or 3% BSA, incubated with primary antibodies for 1 h or overnight, and incubated with peroxidase-conjugated secondary antibodies for 45 min. Bound antibodies were detected using enhanced chemiluminescence (PerkinElmer, Waltham, MA). Semiquantitative densitometric analysis of anti-ezrin and anti-moesin immunoblots from three independent experiments was performed using ImageJ software (National Institutes of Health, Bethesda, MD). The values for ezrin and moesin protein levels were normalized using β-actin as a loading control.
RNA was extracted from NMuMG cells using the RNeasy Mini Kit (Qiagen, Valencia, CA), and first-strand cDNA was synthesized from total RNA using iScript reverse transcriptase (Bio-Rad Laboratories, Hercules, CA). cDNA was amplified using iQ SYBR Green Supermix (Bio-Rad Laboratories) and detected on a CFX96 Real-Time PCR detection system (Bio-Rad Laboratories). Quantitative analysis of ezrin, moesin, and radixin gene expression from at least three independent experiments was performed using CFX Manager software (Bio-Rad Laboratories) and the ribosomal protein gene Rpl19 for normalization. Primers specific for mouse ezrin, moesin, and radixin cDNA were obtained from Qiagen (NM_009510, NM_010833, and NM_009041, respectively). The data were statistically analyzed using one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison post test (Prism 4; GraphPad Software, La Jolla, CA).
Immunolabeling and image acquisition
NMuMG cells grown on glass coverslips were washed three times with PBS at room temperature, fixed with 4% formaldehyde in PBS for 12 min, permeabilized with 0.5% Triton X-100 in PBS for 10 min, and then blocked with 3% BSA in PBS for 30 min or overnight. Fixed cells were incubated with primary antibodies for 1 or 2 h, washed with PBS, and incubated with fluorophore-conjugated secondary antibodies for 45 min. Fixed cells were also incubated with rhodamine-conjugated phalloidin (Invitrogen) for 10 min to stain F-actin and with Hoechst 33342 (Invitrogen) for 10 min to stain nuclei. For plasma membrane labeling, cells were incubated with 4 μg/ml Oregon Green 488–conjugated wheat germ agglutinin (Invitrogen) in PBS for 10 min at 37°C prior to fixation. Coverslips were mounted on slides with ProLong Gold antifade reagent (Invitrogen). Cells were imaged using a 63× Plan-Apochromat/1.40 or a 40× EC Plan-Neofluar/1.30 oil immersion objective on an inverted laser-scanning confocal microscope (LSM510 META; Carl Zeiss, Thornwood, NY), and images were captured using Zeiss software (LSM 510 Meta 4.2 Software). Z-Series projections represent confocal images combined from 16 optical sections acquired at 0.3-μm intervals.
Quantification of elongated cell morphology
Measurements of TGF-β–treated NMuMG cells were made using images of cells that were stained for F-actin and nuclei and were acquired using a 40× objective. The lengths of the major and minor cell axes were measured using Zeiss software (Zeiss LSM Image Browser). The ratios of the major axis to the minor axis of cells were used to determine the degree of elongated cell morphology. For each experiment, between 30 and 40 cells of each cell type were measured. The data were statistically analyzed using one-way ANOVA followed by Dunnett's multiple comparison post test (Prism 4).
Spinning disk confocal and time-lapse microscopy
NMuMG cells grown on glass coverslips were imaged at 37°C using a 40× Plan-fluor ELWD/0.6 air objective (phase contrast) or a 60× Plan Apochromat TIRF/1.45 oil immersion objective (fluorescence) on an inverted microscope system (Nikon Eclipse TE2000 Perfect Focus System; Nikon Instruments, Melville, NY), equipped with a spinning-disk confocal scanner unit (CSU10; Yokogawa, Newnan, GA), a 488-nm solid-state laser (LMM5; Spectral Applied Research, Richmond Hill, Canada), multipoint stage (MS-2000; Applied Scientific Instruments, Eugene, OR), a CoolSnap HQ2 cooled charge-coupled device (CCD) camera (Photometrics, Tucson, AZ), and camera-triggered electronic shutters controlled with NIS-Elements Imaging Software (Nikon). For short-term videos, cells were imaged after 48 h of TGF-β treatment in medium supplemented with 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.5, and images were captured every 1 min. For long-term videos, cells were imaged after 1 h (phase contrast) or 6 h (fluorescence) of TGF-β treatment in medium supplemented with 10 mM HEPES, pH 7.5, in 5% CO2, and images were captured every 10 min. Images for presentation in figures and videos were processed with a 2 × 2 Gaussian low-pass filter to reduce high-frequency pixel noise, and an unsharp mask filter (7 × 7 kernel size; scaling factor 0.5 for short-term videos, or 13 × 13 kernel size; scaling factor 0.7 for long-term videos) to enhance dim features using NIS-Elements Imaging Software.
In vitro wound-healing migration assays and time-lapse microscopy
Monolayers of NMuMG cells grown in six-well plates were wounded using a plastic pipette tip 48 h after the initiation of TGF-β treatment, washed twice with serum-free medium, and replenished with fresh medium. Cells were imaged at 37°C in 5% CO2 using a 10× Hoffmann modulation objective on a Zeiss Axiovert S-100 microscope. Images were captured every 15 min, beginning immediately after wounding and ending 20 h after wounding, using a Spot RT Slider cooled CCD camera (2.3.0; Diagnostic Instruments, Sterling Heights, MI) operated with Openlab software (Improvision, PerkinElmer). Wound-area measurements were determined using ImageJ software. The area of a single wound was calculated as the average of three different cell-free areas from the same wound. The migratory rates were determined by the total decreased wound area from 0 h to 20 h after wounding. For each condition, wounds from four independent experiments were measured. The decreased wound areas for each time point were statistically analyzed using one-way ANOVA followed by Newman–Keuls multiple comparison post test (Prism 4).
Matrigel Transwell invasion assays
NMuMG cells grown in the presence of TGF-β for 48 h were resuspended in DME medium supplemented with 0.2% FBS and were seeded in the upper chamber onto rehydrated Growth Factor Reduced Matrigel Matrix–coated inserts (BD Biosciences, San Diego, CA). The lower chamber was filled with DME medium supplemented with 10% FBS, and the invasion chambers were incubated for 21 h at 37°C in 5% CO2. Noninvading cells were removed from the upper surface of the membrane with a cotton-tipped applicator. Cells were fixed with methanol for 5 min at −20°C, and nuclei were stained with Hoechst 33342 (Invitrogen) for 5 min. Membranes were mounted onto glass slides with Fluorescence Mounting Medium (Dako, Glostrup, Denmark). Cell nuclei were imaged using a 10× Plan-Neofluar/0.3 air objective on a Zeiss Axiophot epifluorescence microscope, and images were captured using a CoolSnap HQ2 camera operated by Micro-Manager software (University of California, San Francisco). Cells were counted using ImageJ software. The number of cells invading per field of view for one membrane was calculated as the average of seven different fields of view from the same membrane. For each cell type, a total of at least five membranes were counted from three independent experiments. The data were statistically analyzed using one-way ANOVA followed by Bonferroni's multiple comparison post test (Prism 4).