Three-dimensional MCF10A assay
Stably transfected MCF10A-EcoR cells were passaged, and subjected to 3D assays in GFR Matrigel and indirect immunofluorescence as previously described [12
]. Acini were incubated with primary antibodies (anti-Deaf-1 polyclonal antisera; diluted 1:50, anti-Ki67; diluted 1:300; Novo Castra) overnight at 4°C. Secondary antibody (Alexa Fluor®
-conjugated anti-rabbit-488; Molecular Probes, Invitrogen, Carlsbad, CA, USA) was diluted 1:200. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI; Sigma, St Louis, MO, USA).
Generation and analysis of Deaf-1 transgenic mice
A fragment encompassing an HA-tagged Deaf-
1 cDNA was cloned downstream of a MMTV-LTR vector which also contained the simian virus 40 (SV40) untranslated (UTR) and poly(A) sequences [10
]. The MMTV-HA-Deaf-1
plasmid was digested with SalI
to release the MMTV-HA-Deaf-1-SV40
fragment from the vector, and subsequently isolated by gel electrophoresis in a 0.7% low melting point agarose gel. The 7 kb fragment was microinjected into the pronucleus of FVB/N fertilised eggs, and these were then transferred to the oviducts of foster mothers (FVB/N). Genomic (tail) DNA from the founder mice was genotyped by PCR. Eleven founder mice were positive for the transgene and mated with wild-type FVB/N mice to obtain F1 generations. All animal experiments were conducted according to the WEHI Animal Ethics Committee guidelines.
Mammary glands were collected from adult female mice at different developmental time-points. Timed pregnancies were scored by observation of vaginal plugs, and confirmed by examination of embryos at the time of mammary gland collection. Lactation time-points corresponded to days post-parturition. Pups were removed after 10 days to initiate involution. The estrous cycle stage of virgin females was determined by vaginal smears, which were dried and stained with haematoxylin and eosin.
Genomic tail DNA from founder mice was digested with HindIII, separated by electrophoresis, transferred to Hybond N+ membranes (Amersham Biosciences, Buckinghamshire, England) and then hybridized to a 1.2 kb SV40 fragment labeled with [α-32P]-dCTP, using the DECAprime™II random primed DNA labeling kit (Ambion, Austin, TX, USA), according to the manufacturer's instructions.
Histological sections and mammary gland wholemounts
For histological sections, portions of the inguinal mammary gland were harvested and fixed overnight in 4% (w/v) PFA in PBS, pH 7.4, at 4°C. Mammary glands were then embedded in paraffin, and 1.5 μm sections stained with haematoxylin and eosin. For wholemount analysis, whole thoracic and inguinal mammary glands were fixed overnight in Carnoy's solution (60% ethanol, 30% chloroform, 10% acetic acid) before staining with haematoxylin.
Lysate preparation, immunoprecipitation and Western blot analysis
Protein lysates from adult mouse organs and mammary epithelial cell cultures were prepared in KALB lysis buffer (150 mM NaCl; 1 mM EDTA; 50 mM Tris.HCl, pH 7.5;10 mM NaF; 1 mM Na3 VO4; 1% Triton X-100)containing Complete protease inhibitor cocktail (Roche, Mannheim, Germany). For immunoprecipitation, 2 μl of anti-Deaf-1 polyclonal antisera was added to 500 μg of protein in a final volume of 200 μl KALB lysis buffer. Samples were incubated on ice for 2 hours before incubation with protein G sepharose beads, washing and Western blot analysis.
Tissue protein lysates (30 – 40 μg of total protein), whole cell lysates (10 – 25 μg of total protein) or immunoprecipitates were resolved on 4–20% Tris-glycine Novex pre-cast polyacrylamide gels (Invitrogen, Carlsbad, CA, USA) and transferred to Immobilon-P polyvinylidene difluoride membranes (PVDF; Millipore, Bedford, MA, USA) using the Novex transfer apparatus. Membranes were then probed with the following primary antibodies: anti-Deaf-1 rabbit polyclonal antisera which was raised against a 16-mer KLH-conjugated peptide (MEDSDSAAKQLQLAEC) located in the amino-terminal of murine Deaf-1 (also recognizes human DEAF-1) diluted 1:1,000, anti-HA rat monoclonal (3F10; Roche) diluted 1:750, and anti-tubulin mouse monoclonal (B-5-1-2; Sigma) diluted 1:5,000.
Bromodeoxyuridine (BrdU) immunodetection
Mice were injected with 0.5 mg/10 g body weight 5-bromodeoxyuridine (BrdU) Cell Labeling Reagent (Amersham Biosciences, Buckinghamshire, England) 1 hr prior to tissue collection. For immunohistochemical detection of BrdU-labeled cells in mouse mammary glands, sections were treated with 3% hydrogen peroxide and permeabilized with 20 μg/ml proteinase K (Sigma, St Louis, MO, USA) followed by 0.2% Triton X-100. They were then incubated sequentially with rat anti-BrdU antibody (BD Biosciences, Bedford, MA, USA), biotinylated rabbit anti-rat IgG secondary antibody (Dako Cytomation, Carpinteria, CA, USA), and HRP-conjugated strepdavidin (LSAB2; Dako Cytomation), before staining with 3,3'-diaminobenzidine (DAB; Dako Cytomation) and counterstaining with haematoxylin. For quantification of proliferating cells within the mammary glands of transgenic and wild-type mice, greater than 1,000 epithelial nuclei in 10 random fields (×400 magnification) were counted.
Immunohistochemistry of mouse mammary sections
Sections were deparaffinized, rehydrated and subjected to antigen retrieval by boiling in 10 mM citrate buffer, pH 6.0 for 20 min, before blocking in 10% normal goat serum (NGS). The primary antibodies, anti-PRa6 (anti-PRB) and anti-PRa7 (anti-PRA), kind gifts from C. Clarke and D. Graham, were diluted 1:40 and 1:80 respectively and incubated overnight at 4°C. For immunofluorescence, sections were incubated with anti-mouse-Alexa Flour®-594 (Molecular Probes, Invitrogen, Carlsbad, CA, USA), mounted with Fluorescent Mounting Medium (Dako Cytomation, Carpinteria, CA) and visualized by fluorescence microscopy. For immunohistochemistry, sections were incubated with biotinylated anti-mouse IgG secondary antibody (Vector Laboratories Inc, Burlingame, CA, USA) diluted 1:500. The tertiary step, counterstaining and quantification were carried out as described above.
Generation of mouse mammary epithelial cells and immunofluorescence
Mammary glands were harvested from 8 week-old female Deaf-1-/-
mice and digested to obtain primary mammary epithelial cells (MECs). MECs were immortalized with an ecotropic retrovirus encoding human papilloma virus (HPV16) E6/E7 proteins as described in [32
To analyze Deaf-1 expression, cells were plated on coverslips at 0.5 × 106 cells/plate in 6-well plates, infected with control or Deaf-1 retrovirus and then selected in puromycin (1.5 μg/ml) for 48 – 72 hr. Cells were incubated with anti-Deaf-1 rabbit polyclonal antibody before incubation with the anti-rabbit-FITC secondary antibody. Nuclei were counterstained with 4',6-Diamidino-2-phenylindole (DAPI) and visualized by fluorescence microscopy.
RNA was purified using the QIAGEN RNeasy Kit (Qiagen, Hilden, Germany) and quality assessed by spectrophotometry and gel electrophoresis before hybridization to Affymetrix slides (GeneChip®
Mouse Expression Set 430 2.0). Quality assessment for Affymetrix arrays is reviewed in [33
] and was carried out in the Bioinformatics Department (WEHI, Melbourne, Australia). Gene symbols were obtained from the Affymetrix probe-set annotation file, version 25, 19 March 2008.
RT-PCR and Quantitative RT- PCR analysis
PCR was performed using 1 μl of cDNA and 50 ng of forward and reverse primers (Sigma-Genosys, Sydney, Australia; see Table ). PCR conditions were as follows: 94°C for 2 min, followed by 30–35 cycles of denaturation at 94°C for 45 sec, annealing at x°C (see Table ) for 45 sec and extension at 72°C for 45 sec, followed by a final extension at 72°C for 5 min.
Primers for RT-PCR and quantitative real-time-PCR analysis
Quantitative RT-PCR assays were performed in a Rotor-Gene™ 6000 (Corbett Research, Mortlake, NSW, Australia) using 2 μl of cDNA in a 20 μl reaction volume containing 50 ng of each primer (Table ) and 1 × SensiMix Plus SYBR®Green I Master reaction mix (Quantace Ltd, London, UK). The amplification program included an initial denaturation step at 95°C for 10 min, followed by 40 cycles of denaturation at 94°C for 15 sec, annealing at 60°C for 30 sec, and extension at 72°C for 30 sec. The 18S rRNA and CK18 analyses were used for normalization to enable construction of standard curves. Relative concentrations were calculated using the delta CT method on the Rotor-Gene™ 6000 software.