Cell Culture and Transfection
R– cells are an embryonic fibroblast cell line derived from mice with a targeted disruption of the IGF-IR, and R+ cells are R– cells that were transfected to express the IGF-IR (Sell et al., 1994
). R+, R–, HeLa, and MCF-7 cell lines were maintained in DMEM (BioWhittaker UK, Berkshire, United Kingdom) supplemented with 1 mM l
-glutamine, 10% fetal bovine serum (FBS), and antibiotics. MCF10A cells were maintained in DMEM/Ham's medium (Sigma Ireland, Dublin, Ireland) supplemented with 5% horse serum, 1 μg/ml insulin, 20 ng/ml EGF (PeproTech, London, United Kingdom), 100 ng/ml cholera toxin, 0.5 μg/ml hydrocortisone, and 2 mM l
-glutamine. For IGF-I stimulations, MCF-7 and MCF10A cells were starved of serum for 24 h before the addition of 100 ng/ml IGF-I (PeproTech) for the indicated times. For culturing cells in suspension, 2 × 106
cells were seeded onto nontissue culture plates that had been coated with poly-HEMA (6 mg/ml) (Sigma Ireland).
For transient transfections of HeLa cells with green fluorescent protein (GFP)- or hemagglutinin (HA)-tagged Mystique isoforms, cells were transfected with 4 μg of DNA by using LipofectAMINE Plus (Invitrogen, Breda, The Netherlands). To generate stable transfectants, MCF-7 cells were transfected with relevant plasmids, and 24 h after transfection cells were cultured in medium containing G418 (Geneticin; 1 mg/ml) for 14 d, at which time individual clones were selected, expanded, and screened for expression of HA-Mystique isoforms by Western blotting.
RNA from 5 × 106 cells was extracted using the TRIzol reagent (Invitrogen) according to the manufacturer's instructions and separated by denaturing formaldehyde gel electrophoresis and transferred to nylon membranes. Prehybridization and hybridization were carried out at 42°C in 50% formamide, 5× SSC, 4× Denhardt's solution, 0.1% SDS, and salmon sperm DNA (100 μl/ml; Sigma Ireland) for 2 and 15 h, respectively. 32P-Labeled probes (>1 × 106 cpm/ml) were prepared by the random primer method (NEBlot, New England Biolabs, Hertfordshire, United Kingdom). Filters were washed twice at 42°C in 2× SSC, 0.1% SDS for 5 min., and then twice at 42°C in 0.1× SSC, 0.1% SDS for 15 min, and exposed to PhosphorImager screens for empirically determined times.
Molecular Cloning of Mystique
Mystique 2 was amplified by reverse transcription-polymerase chain reaction (RT-PCR) on total RNA extracted from MCF-7 cells by using the following primers: MF 5′-cttctcgaggtatggcgttgacgg-3′ and M2R 5′-catctcgagctcaggcccgagag-3′. Two distinct products of ~1.0 and ~0.9 kb were amplified, purified, and cloned using XhoI (bold sequences in primers) into pcDNA3-HaX and pEGFP-C1 (BD Biosciences, Oxford, United Kingdom). Sequencing of inserts confirmed the larger insert (1.0 kb) to be Mystique 2 and the smaller insert (0.9 kb) to be two splice variants of the same size, which we designated Mystique 3a and Mystique 4 (see Supplemental Figures 1 and 2). Mystique 1 was cloned after polymerase chain reaction (PCR) amplification with primers MF (as described above) and M1F 5′-agactcgagcacaccagcttggc-3′ on human cDNA clone FLJ00106 (kindly provided by the Kazusa DNA Research Institute, Chiba, Japan). Mystique 2 L80K, CC313-316, and double mutants were generated by PCR by using the PvuII (L80K) and KpnI (CC313-316SS) restriction sites within Mystique 2 and then cloned XhoI/XhoI into pcDNA3-HaX and pEGFP-C1 vectors.
A Mystique restriction fragment encoding amino acids 1–184 (including the PDZ domain) was cloned into pGEX-6P1 prokaryotic expression vector (Pfizer, Inc., Täby, Sweden). Glutathione S-transferase (GST)-fused 1–184 protein was purified by affinity chromatography and injected into a New Zealand White rabbit (Biological Services Unit, UCC, Cork, Ireland). Affinity-purified polyclonal antibodies were obtained by adsorption to nitrocellulose-immobilized GST-fused 1–184 fragment, elution with 500 μl of 0.2 M glycine, pH 2.15, neutralization with 200 μl of 1 M K2HPO4, pH 7.0, and extensive dialysis against 1× phosphate-buffered saline at 4°C.
Antibodies and Immunofluorescence
Mouse anti-paxillin and anti-phosphotyrosine antibodies were purchased from Upstate Biotechnology (Milton Keynes, United Kingdom). Mouse anti-α-actinin (clone BM75.2) and anti-β-actin antibodies were from Sigma-Ireland. Mouse anti-β1-integrin (clone 12G10) was from Serotec (Oxford, United Kingdom). Mouse anti-HA (clone 16B12) was from Babco (Berkeley, CA).
For immunofluorescence, glass coverslips were coated with 10 μg/ml collagen I (Sigma Ireland) at 4°C overnight. Cells were then allowed to attach onto precoated coverslips for at least 12 h, rinsed with PHEM (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, and 2 mM MgCl2, pH 6.9), fixed in 3.7% formaldehyde in PHEM for 10 min, and permeabilized with 0.1% Triton X (TX)-100 in PHEM for 5 min. After preblocking with 5% normal goat serum (Sigma Ireland) in PHEM for 30 min, cells were incubated with primary antibody, washed with PHEM, and incubated with Cy2- or Cy3-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Soham, Cambridgeshire, United Kingdom). For the detection of F-actin, cells were treated with tetramethylrhodamine B isothiocyanate (TRITC)-phalloidin (Sigma Ireland).
Fluorescence was monitored using a 100× Plan Fluor objective on a Nikon Eclipse E600 microscope. Images were taken with a SPOT camera and adjusted using Adobe Photoshop or MetaMorph software.
Western Blotting and Immunoprecipitation
Whole cell lysates were prepared by lysing cells in ice-cold SDS-lysis buffer (1% Nonidet P-40, 0.1% SDS, 20 mM Tris, 50 mM NaCl, 50 mM sodium fluoride, 1 μM pepstatin, 1 mM phenylmethylsulfonyl fluoride, 1 μM aprotinin, and 1 mM sodium orthovanadate, pH 7.6). Cell debris was removed by centrifugation at 15,000 × g at 4°C for 15 min, and samples were then denatured by boiling in 5× SDS-PAGE sample buffer for 5 min. Detergent-soluble fractions were prepared by lysing cells in ice-cold CSK extraction buffer (10 mM PIPES, pH 6.8, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, and 1 mM EGTA) with 0.5% TX-100 and protease inhibitors. Detergent-insoluble material was pelleted by centrifugation and pellets were resuspended in 2% SDS, 50 mM Tris, pH 7.5.
Proteins were resolved using 4–20% gradient SDS-PAGE and transferred to nitrocellulose membranes (Schleicher & Schuell, Dublin, Ireland), which were blocked with 5% milk in Tris-buffered saline (TBS)-T (20 mM Tris, 150 mM NaCl, and 0.05% Tween 20, pH 7.6) for 1 h at room temperature. Antibodies were diluted in TBS-T, 5% milk and incubated at 4°C overnight. Horseradish peroxidase-conjugated secondary antibodies (DakoCytomation Denmark A/S, Glostrup, Denmark) were used for detection using chemiluminescence with the enhanced chemiluminescence reagent (Amersham Biosciences UK, Little Chalfont, Buckinghamshire, United Kingdom).
For immunoprecipitation of endogenous α-actinin or transfected HA-tagged Mystique 2 mutants, protein extracts were precleared using bovine serum albumin (BSA)-coated protein G Agarose beads (15 μl of beads per 400 μg of total protein in 700 μl of lysis buffer) by incubation at 4°C for 1 h with gentle rocking. The lysates were recovered from the beads by centrifugation at 1000 × g for 3 min and transferred to fresh tubes for incubation with 20 μl of protein G Agarose beads plus primary antibody (3 μg of each antibody) overnight at 4°C with gentle rocking. The beads were washed (3 times) with ice-cold lysis buffer and then removed from the beads by boiling for 5 min in 20 μl of 2× SDS-PAGE sample buffer for electrophoresis and Western blot analysis.
Proliferation and Soft Agar Assays
To measure proliferation in monolayer culture, MCF-7 stable transfectants were cultured in DMEM/10% FBS at 4 × 104 cells per well in multiple wells of a 24-well plate. At intervals, cells were removed from quadruplicate wells and counted by trypan blue exclusion using a hemocytometer.
Anchorage-independent growth was determined by assaying colony formation in soft agarose. MCF-7 cells (103/well) were resuspended in 0.33% low-melting point agarose (Sigma Ireland) in DMEM/10% FBS and plated in triplicate onto 35-mm dishes containing a 2-ml base agarose layer (0.5%). Every 3–4 d, 200 μl of DMEM/10% FBS was added. After 14 d, colonies were stained with 0.01% crystal violet.
Plates (96-well) were coated with either collagen I (10 μg/ml) or fibronectin (5 μg/ml) at 4°C overnight and then blocked with 2.5% BSA for 2 h at 37°C. Cells were starved from serum for 4 h before harvesting with trypsin/EDTA, washing twice with serum-free media (SFM) and then resuspending in SFM 0.01% BSA (DMEM/BSA). The final cell number was determined, and 2 × 104cells per well were resuspended in DMEM/BSA and plated onto collagen I- or fibronectin-coated plates. Cells were allowed to attach at 37°C for the times indicated, and unbound cells were removed by gentle washing with DMEM/BSA. Attached cells were then fixed with methanol and stained with 0.1% crystal violet, which was extracted after extensive washing with 50 μl of 0.5% TX-100 (in water) at room temperature. Incorporated crystal violet was measured by reading its absorbance at A595.
Small Interfering RNA (siRNA) Construction and Transfection
siRNAs targeted to human and mouse Mystique were obtained from Dharmacon (Lafayette, CO) with the following sequences: human Mystique, 5′-aagauccgccagagccccucg-3′; and mouse Mystique, 5′-aagauccgacagagcgccuca-3′ (corresponding to nucleotides 199–219 after the start codon for both human and mouse Mystique). Nucleotides typed in bold indicate where the mouse siRNA differs from the human. MCF-7 and MCF10A cells (30–50% confluent) were transfected with 20 pmol of siRNA by using the Oligo-fectAMINE transfection reagent (Invitrogen) according to the manufacturer's instructions. Protein expression was assessed by Western blotting with the anti-Mystique antiserum from 48 to 96 h after transfection. Adhesion and transwell migration assays were performed 60 h posttransfection.
Transwell Migration Assays
siRNA-transfected MCF-7 and MCF10A cells were starved of serum for 4 h before harvesting with trypsin/EDTA, washing twice with SFM, and then resuspending in DMEM/BSA. The final cell density was determined using a hemocytometer, and 2.5 × 104 cells were resuspended in 100 μl of DMEM/BSA. Lower wells were loaded with either DMEM/bovine serum albumin (control) or DMEM/BSA 10% FBS. After 4 h (MCF10A) or 18 h (MCF-7) at 37°C, chambers were disassembled and cells on the upper surface of the membrane were removed by scraping so that only cells that had migrated through the membrane remained. The membrane was then fixed with methanol, stained with 0.1% crystal violet, washed extensively with water, and air-dried. Cell counts were obtained by averaging cells from five fields at 10× magnification per well from triplicate wells.