Cell culture and the construction of CTGF Tet-off cell line
HC11 mouse mammary epithelial cells, provided by Dr. Nancy Hynes, were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 5 μg/ml insulin, 10 mM HEPES and 10 ng/ml EGF as described [13, 35]. The HC11 cell line containing the pTet-Off plasmid (Clontech, Mountainview, CA) was infected with a pREV-TRE (Clontech) retroviral vector encoding epitope-tagged CTGF/CCN2 and used to express HA-CTGF/CCN2 in response to removal of doxycycline (Dox) as described previously . The HC11-TRE vector control cells and HC11-TRE-CTGF cells were grown in doxycycline (2 μM) which was removed for 96 hours to induce CTGF/CCN2 expression for experiments.
Prior to stimulation with lactogenic hormones, HC11 cells and HC11-derived cell lines were grown to confluence and maintained for 3-5 days to establish competence [9, 39] then maintained in media without EGF for 24 hours. To induce lactogenic hormone-induced differentiation, the cells were rinsed twice and then incubated in differentiation media, RPMI with dexamethasone (10-6M), 5 μg/ml insulin, and 10 μg/ml prolactin, referred to as DIP. Lactogenic differentiation was characterized by analysis of β-casein transcription and by the formation of domed structures referred to as mammospheres, which were visualized by phase contrast microscopy and manually enumerated.
MCF10A acinus formation
Acinus formation was performed with MCF10A cells using an established technique with minor modifications . Growth factor reduced Matrigel was added to wells of an eight-well glass chamber slide. Basement membrane was allowed to solidify for 15 minutes at 37°C. MCF-10A cells were harvested, washed in DMEM/F12, 2% horse serum and resuspended in assay media (DMEM/F12, 2% horse serum, 0.5 μg/ml hydrocortisone, 100 ng/ml cholera toxin, 10 μg/ml insulin). A mixture of assay media containing EGF (20 ng/ml) plus 4% Matrigel was prepared with or without CTGF/CCN2 (25-100 ng/ml, Cell Sciences, Canton, MA). A 1:1 mixture of the Matrigel-containing assay media and MCF-10A cells (25,000 cells/ml) was plated on top of the solidified basement membrane. The cells were incubated at 37°C for 20 days with media changes every 4 days. Each well was fixed with 2% paraformaldehyde followed by permeabilization in 0.5% Triton X-100 at 4°C. Wells were then rinsed with PBS containing glycine (100 mM) and blocked with IF buffer (7.7 mM sodium azide, 0.1% BSA, 0.2% Triton X-100, 0.05% Tween-20, 10% FBS). The cells were washed in PBS and mounted to slides with ProLong Gold antifade reagent containing DAPI (Molecular Probes, Invitrogen, Eugene, OR). Confocal imaging was performed on a Zeiss Pascal Laser Scanning Confocal Microscope (Carl Zeiss, Thornwood, NY).
RNA isolation, RT-PCR, northern blotting, and Southern blotting
Total RNA was isolated and purified using the TriPure reagent (Roche, Palo Alto, CA). Northern blots were prepared with 10 μg of total RNA separated on a 1% agarose-formaldehyde gel and transferred to a nylon filter. Reverse transcriptase PCR (RT-PCR) was performed using the GeneAmp RNA PCR Kit and primers for β-casein amplification as follows: Fwd 5' CATCCTTTCAGCTTCACC, Rev 5' AGAGACAGCTGGGTCTGAG. Primers for mouse β-actin were as follows: Fwd 5' CTAAGGCCAACCGTGAAAAGA, Rev 5' GAGGTCTTTACGGATGTCAAC. PCR products were electophoresed on a 1% agarose gel. The DNA was denatured and neutralized and transferred to a nylon filter. Northern and Southern blots were hybridized as described previously . The probes for CTGF, β-casein and actin have been described [27, 37].
Confluent HC11-TRE and HC11-TRE-CTGF cells were stimulated with DIP for 16 hours prior to cross-linking with 1% formaldehyde for 10 minutes at room temperature, followed by treatment with 1.12 ml of 2.5 M glycine for 5 minutes at room temperature. The precipitating antibodies used included anti-rabbit IgG (Chemicon, PP64) or anti-Stat5 N-term (Santa Cruz, sc-836). Primers for the β-casein proximal promoter, forward 5'CCAGCTTCTGAATTGCTGCC3', reverse 5' GGTCTATCAGACTCTGTGAC3', were used in PCR reactions of 35 cycles of 1 minute at 95°C, 30 seconds at 55°C, 30 seconds at 72°C. Amplified DNA was analyzed by electrophoresis on an ethidium bromide stained 1.5% agarose gel. Bands were quantitated using the Fujifilm LAS-4000 (Fujifilm Medical Systems USA, Inc.). Results are expressed as a ratio of immunoprecipitated DNA to the input DNA.
Western blotting and Immunoprecipitation
The procedures for western blotting have been described [27, 37, 38, 55]. The antibodies used in this study include: mouse anti-β-actin (Sigma, St. Louis, MO), goat anti-CTGF (Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-β1 integrin (BD Transduction, BD Biosciences, San Diego, CA), mouse anti-FAK (BD Transduction), rabbit anti-phospho-FAK (Upstate Technologies), rabbit anti-Akt (Cell Signaling Technolgies, Danvers, MA ), rabbit anti-phospho-Akt (Cell Signaling Technology), rabbit anti-Bcl-xL (Santa Cruz Biotechnology), mouse anti-PINCH1 (BD Transduction), rabbit anti-ILK (Santa Cruz Biotechnology), rabbit anti-actopaxin/parvin (Sigma), mouse anti-p130CAS (Upstate Technologies), mouse anti-paxillin (BD Transduction), rabbit anti-c-src (Santa Cruz Biotechnology), mouse anti-vinculin (Sigma). The rabbit anti-Rsu-1 antibody has been described .
To detect focal adhesions, HC11-TRE and HC11-TRE-CTGF cells were grown in the absence of doxycycline for 4 days, at which time they were seeded on coverslips and maintained in serum-free media + EGF (10 ng/ml) for 4 days. The cells were washed with PBS, fixed in 4% paraformaldehyde then permeabilized with 0.5% Triton X-100 and blocked in 4% BSA prior to an overnight incubation at 4°C with an anti-vinculin antibody (Sigma). Coverslips were washed with PBS and incubated for 30 minutes in the dark at room temperature with an AlexaFluor 488-conjugated secondary antibody then mounted with ProLong Gold antifade reagent containing DAPI (Molecular Probes, Invitrogen, Eugene, OR). Immunofluorescence was visualized using an Olympus IX71 microscope, a QImaging Retiga 2000RV camera, and QCapture Pro 6.0 software (Olympus America Inc, Center Valley, PA).
MTT and TUNEL Assays
Proliferation of HC11-TRE-CTGF and vector control HC11-TRE cells was determined by MTT assay (CellTiter96 Assay, Promega, Madison, WI). Viable cells grown in the absence of doxycycline for 96 hours were replated at a density of 1.5 × 104 per well in quadruplicate wells of a 96-well plate in serum-free media with EGF (10 ng/ml). HC11-TRE cells were analyzed following exposure to conditioned media from HC11-TRE-CTGF cells, and in the presence of RGD- or RAD-containing peptide (500 μM, BioMol, Plymouth Meeting, PA). The cells were incubated for 24, 48, 72, or 96 hours. Analysis of the MTT assay has been described previously .
Apoptosis was detected in vector control HC11-TRE cells and HC11-TRE-CTGF cells by TUNEL technology (In Situ Cell Death Detection Kit, Fluorescein, Roche). Cells previously grown in the absence of doxycycline for 96 hours were grown on coverslips in serum-free media in the presence of EGF (10 ng/ml) for 96 hours. The cells were fixed in 4% paraformaldehyde, permeabilized in 0.1% Triton X-100 on ice, and rinsed in PBS and incubated in 50 μl TUNEL reagent for 1 hour at 37°C in the dark. The cells were rinsed in PBS prior to being mounted on slides (Vectashield mounting media, Vector Labs, Burlingame, CA). Cells were viewed on the Olympus BX61 and analyzed by IVision software (IVision, Atlanta, GA).
Cell cycle analysis and surface level expression of α6 and β1 integrins were determined by flow cytometry. HC11-TRE and HC11-TRE-CTGF cells grown without doxycycline for 96 hours were propagated for 96 hours in serum-free media with EGF (10 ng/ml). The cells were harvested with Cell Stripper (Mediatech, Manassas, VA), pelleted, washed, and counted. For cell cycle analysis, cells were resuspended in PBS and methanol was added dropwise for fixation prior to storage at -20°C for 24-96 hours. The cells were pelleted and resuspended in cold PBS and propidium iodide (50 μg/ml) was added to the cells prior to flow cytometric analysis. For integrin expression analysis, the cells were aliquoted into 1 ml samples of 500,000 cells each and washed in FACS wash buffer (1% FBS, 0.05% sodium azide). Cells were then incubated in buffer with antibodies against β1 integrin (BD Biosciences) or α6 integrin (BD Biosciences) or the isotype controls for 1 hour at 4°C then pelleted and resuspended FACS buffer containing PE- or FITC- conjugated secondary antibodies for 45 minutes at 4°C. The cells were fixed in Cytofix (BD Biosciences) on ice, washed and resuspended in FACS buffer. All flow cytometry was performed using a BD Biosciences LSRII cytometer. Cell cycle analysis was performed using ModFit LT software and integrin expression analysis was performed using WinList software (Verity Software House Inc.).
CTGF/CCN2 adhesion assay
To determine the interaction between CTGF/CCN2 and integrin complexes, HC11-TRE cells were seeded in serum-free media + EGF (10 ng/ml) on maleic anhydride Reacti-Bind microtiter wells (Thermo Scientific, Rockville, MD) coated with recombinant CCN2 protein (Cell Sciences, Canton, MA). The plates were coated with CTGF/CCN2 (2 μg/ml) overnight at 4°C, followed by blocking with 1% BSA for 2 hours at 37°C. HC11-TRE cells were suspended at a concentration of 5 × 105 cells/ml, and where indicated mixed with EDTA (2.5 mM), EDTA (2.5 mM) + Mg2+, blocking antibodies to αV, β3 (Santa Cruz Biotechnologies), α6, or β1 integrins (BD Biosciences), or isotype control antibodies. Cells were allowed to attach for 4 hours at 37°C, washed gently with PBS and fixed with 3.7% formaldehyde for 10 minutes at room temperature. Fixed cells were washed with stained with crystal violet and the adherent cells were quantified by reading the absorbance at 570 nm.