h 2 mg/ml Dox. The media was collected after 48 hours and concentrated using AmiconH Ultra 3K Centrifugal Filter Units for protein analysis or treatment of wildtype fibroblasts. Wildtype fibroblasts were grown to 80% confluency in basic growth media, washed twice with PBS, and treated with concentrated conditioned media diluted back to 16strength with minimal media for 48 hours at 37uC and 5% CO2. After 48 hours, the fibroblasts were either harvested for protein analysis by Western blot or gene expression analysis by qRT-PCR. RNA isolation and quantitative RT-PCR RNA was isolated from control and p190B-associated fibroblasts and wildtype fibroblasts from conditioned media and coculture experiments using Trizol and RNeasy RNA purification columns according to the manufacturer’s recommendations. One mg of RNA was converted to cDNA using the RT2 First Strand Kit. Control and p190B-associated fibroblast cDNA was amplified using RT2 Profiler PCR Array Mouse Extracellular Matrix and Adhesion Molecules per manufacturer’s instructions. Data were analyzed using the web-based software RT2 Profiler PCR Array Data Analysis from SABiosciences. The reaction conditions were as follows: 95uC for 10 minutes then 40 cycles of 95uC for 15 seconds and 60uC for 1 minute followed by a melting curve. Col1a1, Fn1, Lama1, and Ctgf mRNA levels were normalized to GAPDH mRNA levels and the data were analyzed using comparative CT. Each experiment was conducted in triplicate and the data represent 23 independent experiments each consisting of 34 animals per genotype pooled. Co-culture experiments Control and p190B overexpressing MECs were CP 868596 plated in basic growth media with 2 mg/ml Dox for 48 hours prior to addition 26646986 of wildtype fibroblasts at a concentration of 2.5:1. The co-cultures were incubated for 7296 hours at 37uC and 5% CO2 in basic growth media with 2 mg/ml Dox and 5 mM SB431542 TGF-b receptor inhibitor in DMSO or DMSO. The fibroblasts were removed by differential trypsinization with a 3-5 minute incubation at 37uC in 0.05% trypsin-EDTA for protein analysis by Western blot or gene expression analysis 
by qRT-PCR. Tissue preparation, immunostaining, and analysis Five-week old control and p190B overexpressing mice were treated for 7 days with Dox diet prior to euthanasia and mammary gland dissection. The number 4 mammary gland pairs were dissected, fixed in 4% PFA, and embedded in paraffin. Five mm sections were cut for histological and immunostaining analysis. The tissues were stained by standard H&E, Masson’s Trichrome Stain kit, or immunostaining to study specific proteins. For immunostaining, the tissues were deparaffinized in xylene and rehydrated through ethanols of decreasing concentration. Antigen retrieval involved microwaving the slides in 10 mM Sodium Citrate, pH 6 for 20 minutes. Five percent BSA plus 0.5% tween-20 in PBS was used as a block and diluent for antibodies. Sections were incubated overnight with primary antibodies and for one hour with secondary antibodies. For immunohistochemistry, Elite ABC Reagent and DAB were used to develop the immunoperoxidase staining and the slides were counterstained with hematoxylin. For immunofluorescence, 26976569 the nuclei were stained with DAPI. Primary antibodies included: Ki67 1:5000; LOX 1:250 no antigen retrieval. Secondary antibodies consisted of anti-rabbit Alexa Fluor 555 and biotinylated anti-rabbit. To quantify stromal thickness on H&E sections, the thickest regions of the stroma were measured at the tip an