We used commercial EHS matrix (Matrigel; Collaborative Research) for the rBM assays; Vitrogen 100 (bovine skin collagen I; Celtrix Laboratories), 3 mg/ml and 10 μg/ml of affinity-purified LM-5 (Russell et al., 2003
) for coating culture dishes; and 0.3% Cellagen Solution AC-5 (ICN Biomedicals) for the 3D collagen I assays. Primary antibodies were as follows: LM-5, rabbit sera pKa1, and clone BM165 (Russell et al., 2003
); α6 integrin, clone GoH3 (BD Biosciences); β1 integrin, clones AIIB2 (provided by C. Damsky, University of California, San Francisco, San Francisco, CA), and TS2/16 (ATCC); β4 integrin, rabbit sera, and clones 3E1, ASC-3, and ASC-8; and α2 integrin, clone 10G11 (all from Chemicon International); IκBα/MAD-3, clone 25, and NFκB p65, rabbit sera (Santa Cruz Biotechnology, Inc.) and clone 20 (BD Biosciences); cytokeratin 18, clone RCK106 (BD Biosciences); RAC1, clone 102 (BD Biosciences); c-myc, clone 9E10 (Oncogene Research Products), and AKT and Phospho-ser472/473/474-AKT; ERK1, rabbit sera (BD Biosciences), phosphoERK1/2 (Thr202/Tyr204), rabbit sera (New England BioLabs, Inc.); activated caspase 3, rabbit sera (Cell Signaling), and HA.11, clone 16B12 (Babco). Secondary antibodies were as follows: horseradish peroxidase, and biotinylated mouse IgG (Vector Laboratories); FITC, and Texas red–conjugated and nonconjugated anti–mouse, anti–rat, and anti–rabbit goat polyclonal antibodies and nonspecific rat and mouse IgGs (Jackson ImmunoResearch Laboratories). Reagents were as follows: NFκB SN50, active cell-permeable inhibitor peptide (50 μM in PBS), NFκB SN50M, inactive cell-permeable control peptide (50 μM in PBS); the EGFR-specific tyrosine kinase inhibitor Tyrphostin AG 1478 (160 μM in DMSO), and the Rho GTPase inhibitor toxin A Clostridium difficile
(10 mM in DMSO; Calbiochem); the MEK1 inhibitor PD98059 (50 μM in DMSO); and the PI 3-kinase inhibitor LY 294002 (50 μM in ethanol; BIOMOL Research Laboratories, Inc.).
The HMT-3522 MECs were grown in 2D and embedded (0.5–0.8 × 106
cells/ml) within ECM gels and phenotypic reversion of T4-2s using β1 integrin mAb AIIB2 or Tyrphostin AG 1478 as described previously (Wang et al., 1998
Cell adhesion was assessed using a fluorescence attachment assay. In brief, plates coated with LM-5 or rBM (100 μg/ml) were blocked (1 h; 0.1% BSA), incubated (60 min, 37°C), washed (3× PBS), incubated with 4 μM calcein (20 min, RT), and quantified using a fluorescence plate reader (model Fluoroskan Ascent Fl; LabSystems).
Anchorage-independent growth was assessed using a soft agar assay (Wang et al., 1998
). In brief, 20,000 cells were plated in 1 ml DME/Ham's F12 containing 0.7% agarose, overlaid with 1 ml of 1% agarose, and 40-μm colonies were scored positive after 21 d.
To inhibit integrin function or LM-5 binding, cells were incubated with mAbs against β1 integrin, clone AIIB2 (1:25–1:100 ascites/ml ECM); β4 integrin, clones ASC-3 or ASC-8 (4–16 μg IgG/ml ECM); LM-5, clone BM165 (10 μg IgG/ml ECM); or IgG isotype-matched control mAb (4–16 μg IgG/ml ECM) at the time of embedment. To inhibit NFκB nuclear translocation, the active inhibitor NFκB SN50 or the inactive analogue NFκB SN50M was added directly to the media.
Cells were directly fixed using 2–4% PFA or 100% methanol, and samples were incubated with primary mAbs, followed by either FITC- or Texas red–conjugated secondary antibodies. Nuclei were counterstained with DAPI (Sigma-Aldrich). Cells were either visualized using a scanning confocal laser (model 2000-MP; Bio-Rad Laboratories) attached to a fluorescence microscope (model Eclipse TE-300 [Nikon] or model MDIRBE [Leica]). Confocal images were recorded at 120× and conventional images were recorded at 40–60×.
Apoptosis was assayed by the Live/Dead Assay (Molecular Probes) or by detection of internucleosomal DNA fragmentation in fixed cells using an in situ TUNEL assay (Boehringer) or via immunodetection of activated caspase 3. Percent death was calculated as cells positive for ethidium bromide expressed as a percentage of the total number of live cells scored positive by calcein staining (FITC). The apoptotic labeling index was calculated as the percentage of total cells positive for FITC-labeled 3′OH DNA ends, and percent apoptosis was determined as the percentage of total cells positive for activated caspase 3. The minimum number of cells scored was 200–400 per experimental condition. Cell death by apoptosis was confirmed by showing that DNA cleavage or caspase 3 activity could be inhibited by prior treatment with the caspase inhibitors YVAD CHO or DEVD-CHO (1 μM; BIOMOL Research Laboratories, Inc.).
Full-length β4 pRK-5 (provided by F. Giancotti, Memorial Sloan-Kettering Cancer Center, New York, NY) was used directly. The 2,710-bp EcoRI–BglII fragment from the β4pRK-5 construct was ligated with the EcoRI–BamHI vector fragment of pEGFP-N2, and an EcoRI–NotI fragment containing the β4 integrin EGFP fusion was subcloned into an EcoRI–NotI vector fragment of Hermes HRS puro-GUS (provided by H. Blau, Stanford Medical Center, Stanford, CA). Myc-tagged V12RAC1 (provided by A. Hall, University College, London, UK) was cloned as an EcoRI fragment into LZRS-IRES-blasticidin; and N17RAC1 and N19RhoA (provided by E. Butcher, Stanford Medical Center) were cloned into the EGFP fusion vector EGFP-C1 (CLONTECH Laboratories, Inc.), and excised and recloned into LZRS-IRES-blasticidin by PCR using the EcoRI tailed primer GTPaseF, 5′. IκBαM and p65 cloned into PLZRS (provided by P. Khavari, Stanford Medical Center) were used directly. The BglII–BamHI fragment containing HA-tagged dominant-negative AKT (K179M) and the HindIII–EcoRI fragment containing the myristoylated HA-tagged AKT (provided by P. Tsichlis, Tufts University, Boston, MA) were subcloned into pLZRS.
Gene expression manipulations
Amphotrophic retrovirus was produced in either modified 293 cells or in Phoenix ampho cells (provided by G. Nolan, Stanford Medical Center), and MECs were spin infected and selected using blasticidin. MECs were transfected with full-length β4 pRK-5 and pcDNA 3.1 plasmid vector DNA or vector plasmid alone using LipofectAMINE (GIBCO BRL), and selected using G418. S-1 β4 pRK-5–transfected cells were enriched for increased membrane localized β4 integrin through differential adhesion to LM-5, and increased β4 integrin levels were verified by FACS® analysis.
Electrophoretic mobility shift assay
To prepare nuclear extracts, cells were washed (1× PBS) and homogenized in nuclear isolation buffer (10 mM Hepes, pH 7.9, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1mM DTT, and 1 mM Pefabloc SC) with an addition of IGEPAL to 0.5%. After incubation (10 min, 4°C), nuclei were isolated by centrifugation (1 min, 14,000 rpm, 4°C) and nuclear extracts were prepared by homogenization and incubation in nuclear extraction buffer (20 mM Hepes, pH 7.9, 420 mM KCl, 1.5 mM MgCl2, 20% glycerol, 0.5 mM DTT, 1 mM Pefabloc SC, and 10 μg/ml leupeptin), followed by centrifugation (15 min, 14,000 rpm, 4°C). Equal amounts of nuclear protein were used in the EMSA reaction with the NFκB consensus oligonucleotide sequence (5′-AGT TGA GGG GAC TTT CCC AGG C-3′). 32P-Labeled oligonucleotide (150,000 cpm) was incubated with 5 μg of nuclear extract and gel shift binding buffer (10 min, RT; Promega Gel Shift Assay System). p65 rabbit antisera were added after the binding reaction, and the mixture was reincubated (20 min, RT). Specificity of binding was tested using competition analyses in which 10-fold molar excess of nonlabeled oligonucleotide sequence was added to a binding reaction. Complexes were resolved in 4.5% polyacrylamide gels (TE buffer: 90 mM Tris, 90 mM boric acid, and 2 mM EDTA, pH 8.0).
Cells were isolated, nonspecific binding was blocked (60 min Dulbecco's PBS, 0.1% BSA) and incubated with saturating concentrations of primary mAb (1 h), washed three times with Dulbecco's PBS, and labeled with FITC-conjugated goat immunoglobulin. Stained cells were washed three times with Dulbecco's PBS and immediately analyzed on a FACScan™ (Becton Dickinson). All manipulations were conducted at 4°C.
Cells were lysed (RIPA buffer: 50 mM Tris-HCl, pH 7.4, 150 mM sodium chloride, 1% NP-40, 0.5% deoxycholate, 0.2% SDS containing 20 mM sodium fluoride, and 1 mM sodium orthovanadate, and a cocktail of protease inhibitors), and equal amounts of protein were separated on reducing SDS-PAGE gels, immunoblotted, and detected with an ECL-Plus system (Amersham Biosciences). To assay for differences in total secreted LM-5, LM-5 was immunoprecipitated from conditioned media, and protein from equal cells was separated on SDS–polyacrylamide gels, immunoblotted, and detected as above.
Cells were treated with vehicle or 20 ng/ml EGF and incubated for indicated times, washed (2× PBS), and extracted (G protein buffer: 25 mM Hepes, pH 7.5, 150 mM NaCl, 1% Igepal CA-630, 10 mM MgCl2, 1 mM EDTA, 10% glycerol, 1 mM Pefabloc SC, 10 μg/ml leupeptin, 10 μg/ml aproptinin, 1 mM sodium orthovanadate, and 1 mM sodium fluoride; 5–10 min). Lysates were centrifuged (10 min, 14,000 rpm), and supernatants were mixed with GST-PBD and incubated with glutathione-Sepharose beads (Amersham Biosciences; 60 min). Lysates were washed (3× lysis buffer), and bound protein was eluted with Laemmli buffer and separated on a 12% SDS–polyacrylamide gel. Active RAC was detected by immunoblotting with anti-RAC antibody, and specific activity was calculated by normalizing densitometric values of PAK-associated RAC to total RAC and E-cadherin. Purified GST-PBD, encoding amino acids 70–117 of PAK1, fused to GST (provided by J. Chernoff, Fox Chase Cancer Center, Philadelphia, PA).