Isolation and culture of HMEC
The mammary epithelial cells (HMEC) were all derived from reduction mammoplasties of individuals with no known breast pathology, as reconfirmed by histopathology of the post-surgical specimens. HMEC were prepared by the method of Smith et al.  and grown at 37°C and 5% CO2 in DFCI-1 media . Cells were allowed to pass through stasis before infection with the hTERT construct, as described by Foster et al., Kiyono et al., and Rothenberg and Nolan .
Cell proliferation and staining
HMEC (human normal mammary epithelial cells) and our tumorigenic cells were seeded in glass 8-well chamber slides at a concentration of 104 cells per well and treated with FTI-277 and DMSO at 2.5, 5.0, 10.0, 15.0 and 20.0 μM concentrations for 5 days in duplicate. Cells were counted using Hemocytometer (blood cell counter) on 1st, 2nd and 5th days as well as stained with Phalloidin-TXR for cytoskeletal staining and DAPI for nuclear staining.
Metaphase chromosomes from HMEC were prepared and G-banded (Wright's stain) using standard cytogenetic techniques . Briefly, after the cells were incubated with 0.06 ug/ul of colcemid at 37°C for 2.5 hours, they were trypsinized with trypsin/EDTA (0.5%/0.1%) and incubated in 2 ml of a 1:1 (by vol) mixture of 0.8% sodium citrate and 0.075 M potassium chloride for thirty minutes at 37 degrees C. This was followed by addition of 1 ml of Carnoy's fixative (3:1 by vol; methanol:acetic acid) and incubation at room temperature. The fixed cells were then spun down and resuspended in 1 ml of Carnoy's fixative. Metaphase chromosomes were prepared for G-banding and Spectral Karyotyping (SKY). Cells were analyzed based on clonality criteria and karyotypes were described by the recommendations of the International System for Human Cytogenetic Nomenclature (ISCN 2005) .
SKY  was used to identify or confirm the G-band karyotype assignments. Briefly, SKY is a form of fluorescence in situ hybridization (FISH) which utilizes a mixture of "whole chromosome paint" DNA probes to uniquely label each of the 24 different human chromosomes, allowing simultaneous visualization and identification of each chromosome or chromosome derivative. The limit of resolution of SKY for structural abnormalities is currently unknown, and the technique will likely not detect subtle, intra-chromosomal rearrangements, such as small deletions, duplications or inversions. We also cannot rule out the possibility of mosaicism for numerical or structural abnormalities given the limited number of cells (usually five), which are evaluated in this study. Unbanded or de-stained G-banded metaphase chromosome preparations were hybridized with the SKY probe mixture and were analyzed using SKYView imaging software, following the manufacturer's instructions (Applied Spectral Imaging, Inc.; Carlsbad, CA). The hybridized metaphase cells were simultaneously stained with the nuclear stain, DAPI. When the bright-dark fluorescent bands from chromosomes stained with DAPI are reversed by the SKYView imaging software, a low-resolution G-band-like pattern is produced that is used to confirm the chromosome identity and to assign cytogenetic breakpoints on rearranged chromosomes.
For both G-band and SKY analyses, structural chromosome abnormalities and chromosome gains were considered clonal if the aberration was exhibited in at least two cells from a culture. Whole chromosome losses were considered clonal if observed in at least three cells from a culture, per ISCN convention .
The protocol as previously described was followed . Cells were lifted from the plate with trypsin/EDTA (0.5%/0.1%) and fixed overnight in 80% ethanol at 4°C. Cells were washed with PBS the following day, and this was followed by staining in PBS with 10 ug/ml of propidium iodide for twenty minutes at room temperature.
Transduction of H-ras 61L into HMEC
pCTV3H H-ras 61L (a generous gift of Dr. Channing Der) or pCTV3H (empty control vector) was precipitated and incubated with Phoenix cells to produce infectious supernatant . Cells were transduced with either construct by incubation with 1 ml of infectious supernatant with 4 pg/ml of polybrene for four hours. Two days later, continuous selection with hygromycin B (20 ug/ml) was initiated over a several week time span until sustained growth from a robust population was maintained.
Soft agar assay
Six-well plates were filled with two ml of 0.66% noble agar in DFCI-1 media as a bottom layer and allowed to solidify at 4°C overnight. 105 cells were suspended in one ml of 0.33% noble agar in DFCI-1 media and plated onto wells. Each well was fed once a week with one ml of 0.33% agar in DFCI-1 media. Wells were scored for colony growth over the next four weeks. Cell clumps greater than 60 um were scored as colonies.
Nude mice injection and tumor harvesting
Four-week-old female nude mice were γ-irradiated to 300 rads using a linear accelerator and injected subcutaneously over the abdomen with 107 cells derived from the soft agar colony that was regrown in monolayer. The mice were observed for tumor formation over the following 30 days. Tumors were harvested and cut into two parts. One part was sent for paraffin embedding, while the other half was minced and disaggregated with trypsin/EDTA (0.5%/0.1%) overnight at 4°C, followed by trypsinization for an additional thirty minutes at 37°C. Single cells were plated and allowed to grow in DFCI-1 media.
Immunohistochemistry on paraffin embedded tumor sections was performed as previously described by Gown and Vogel  at Phenopath Laboratories (Seattle, WA) or the laboratory of Dr. Peggy Porter (Program in Cancer Biology, Fred Hutchinson Cancer Research Center, Seattle, WA) using 4 um thick sections baked onto positively charged slides. Immunohistochemical studies were performed on formalin-fixed, paraffin-embedded, tissue sections using the avidin-biotin-peroxidase complex (ABC) method. Sections were incubated with a cocktail of two anti- cytokeratin mouse monoclonal antibodies (AE1 and AE3, Roche, Indianapolis, IN, 1:200 dilution) that recognizes a wide range of high and low molecular weight cytokeratins; anti-cytokeratin-7 (OV-TL 12/30, DAKO, Carpinteria, CA, 1:1000), anti-smooth muscle myosin heavy chain (SMMS-1, DAKO, 1:30); anti-estrogen receptor (ID5, Zymed, South San Francisco, CA, 1:200); anti-progesterone receptor (IA6, Lab Vision, 1:50); anti-mammoglobin(304-1A5 1;2000, 31A5 1:400, Corixa, Seattle, WA); anti-cytokeratin 5/6(5/16 B4, Chemicon, Temecula, CA, 1:1000); anti-cytokeratin 19 (BA 17, DAKO, 1:100), anti-calponin (Calp, DAKO, 1;1000); anti-cytokeratin 20 (KS 20.8, DAKO, 1:250); anti-cytokeratin 8 (35βH11, DAKO, 1:50); anti-E-cadherin (HECD-1, Zymed, San Francisco, CA,1:300); brst-2 (D6, Signet, Dedham, MA, 1:200). After overnight incubation, slides were incubated with secondary antibody. For polyclonal antibodies raised in rabbit, biotinylated goat anti-rabbit immunoglobulin (1:200) was used. For murine monoclonal antibodies, we used biotinylated goat anti-mouse immunoglobulin (1:200). Sections were incubated in secondary antibody for 30 minutes. The peroxidase staining procedure was done using the ABC Elite kit (Vector laboratories, Burlingame, CA). The immunostaining reaction was visualized using 0.05 % 3,3'- diaminobenzidine in Tris- HCl buffer containing 0.01 % hydrogen peroxide, pH 7.6.
Total cell protein was extracted from transduced cells by sonication in We 16 buffer (25 mM Tris HCl- pH 7.5, 125 mM NaCl, 2.5 mM EDTA, 0.05% SDS, 0.5% NP40, 0.5% DOC, 10% glycerol, 1 mM DTT, 0.5 mg/ml Pefablock, 0.025 mg/ml leupeptin, 0.01 mg/ml pepstatin, 0.01 mg/ml aprotinin, 0.08 M β glycerophosphate, 0.005 Na3VO4, 0.05 M NaF). Twenty micrograms of protein from each cell population was electrophoresed and transferred to a polyvinylidene difluoride membrane (NEN). The membranes were blotted with the following antibodies: H-ras (LA-069, Quality Biotech, Camden, NJ, 1:10,000); c-Myc (9E10, BD Biosciences, San Diego, CA, 1:250), Mdm2 (IF2, Oncogene Research Products, Boston, MA, 1:50), p53 (DO-1, Oncogene Research Products, 1:100), and actin (c-11, Santa Cruz Biotechnology, Santa Cruz, CA, 1:100). Samples destined for H-ras western blotting were run on a 15% SDS polyacrylamide gel, while all other samples were run on 8% polyacrylamide gel. After the samples were transferred onto the membrane, the membrane was blocked in 10% milk in TBST buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween-20) for one hour at room temperature or overnight at 4°C. Membranes were then incubated overnight at 4°C with 10 ml of 5% milk in TBST/0.5% Tween 20 with the primary antibody. The following day, membranes were briefly washed and incubated for one hour with secondary antibody in 5% milk in TBST/0.5% Tween 20 with the appropriate secondary antibody. Anti- mouse IgG- HRP from Jackson Immunoresearch Labs (Bar Harbor, ME) was used against the H-ras (1:35,000), c-Myc (1:10,000), and p53 (1:10,000) primaries. Donkey anti-goat antibody (Santa Cruz Biotechnology) was used against the polyclonal actin primary (1:5,000). Blots were developed using the Renaissance chemiluminescence system (NEN) and imaged on Kodak Xomat blue film.