gly associ ated with breast cancer risk.@ Additional studies are re quired to es

T 3. 0,The experiment was performed twice with six replicates. 3D Matrigel assay Cells were cultured on growth [You must be registered and logged in to see this link.] factor reduced Matrigel using the overlay method,Briefly, 4 chambered Lab Tek chamber slides or 24 well plates were coated with Matrigel. Cells suspended in 2. 5% Matrigel solution were added on coated chamber slides and allowed to grow up to 17 days.@ 3D PEG gel assay MMP degradable polyethylene glycol gel with RGD peptides was purchased from QGel,Briefly, 400 ul of Buffer A was mixed with QGelTM MT 3D Matrix powder, before addition of 100 ul of cell suspension,Drops of 40 ul were applied into a disc caster and after 30 min incubation at 37 C the gelled discs were removed and placed on 24 well plates with 1 ml of medium per well. The cells were allowed to grow up to 18 days.

Immunofluorescence The MCF 10A [You must be registered and logged in to see this link.] cells in Matrigel and PEG gel were fixed in 4% paraformaldehyde for 1 hour at 37 C followed by permeabilization with 0. 1% Triton X100 for 45 min at room temperature and blocking with 3% BSA for 1. 5 hours at 37 C. The fixed cells were incubated with mouse monoclonal anti 6 integrin antibody for 1. 5 hours at 37 C. The secondary goat anti mouse Alexa Fluor 488 was used similarly.@ The cells were stained with DAPI and mounted with Vectashield,Images were taken with Zeiss Axio Imager. M2 microscope connected to an ApoTome slider module,Image analysis Images were taken from the cells in Matrigel and PEG gel using Olympus IX71 microscope and processed with ImageJ,Four images from each experiment at designated time points were analyzed and the average area covered by the cells was calculated.

Protein extraction The cells were collected 24 hours or 5 days and 4 or 7 days after first addition of BMP4. Matrigel was first dissolved by adding cold PBS with 5 mM EDTA and the cells were kept on ice for 15 min. The cell Matrigel solution [You must be registered and logged in to see this link.] was then collected, kept on ice for 30 min and centrifuged for 15 min at 3300 × g, at 4 C.@ Cells were lysed and protein concentration measured as previously described,Western blot Fifty ug of protein was loaded onto SDS PAGE gels. After gel electrophoresis, the proteins were transferred to a PVDF membrane. The following primary antibodies and dilutions were used. p21,Cdk4,Cdc2,p Cdc2,p27,p16,p15,Cyclin B1,Cyclin B2 and Cyclin D1,All antibodies were rabbit polyclonal, with the exception of p16 and Cyclin B2,In addition, a mouse monoclonal anti GTF2H1 antibody was used.

Pro teins were detected using the BM Chemiluminescence Western Blotting kit according to manufacturers instructions. Anti mouse rabbit secondary antibody was used for all antibodies, except for Cyclin B2, which was detected with anti goat secondary antibody,The membranes were stripped and probed with B tubulin as a loading control.@ Quantitative RT PCR The expression of MMP 1, 2, 3, 7, 9, 14 and ADAM17 was examined in BMP4 and vehicle treated MDA MB 231 and BT 474 cells grown for 14 days in Matrigel. The cells were harvested as described above for protein extraction. Total RNA was extracted using RNeasy Mini kit and was reverse transcribed using SuperScriptTM III First Strand Synthesis System for RT PCR as described,qRT PCR was performed using gene specific primers and UPL probes and the LightCycler equipment as described with 1.

2 uM con centration of primers and probes and the following pro gram. 10 min denaturation at 95 C followed by 45 cycles of 10 s denaturation at 95 C, 10 s annealing at 55 C and 15 s elongation at 72 C. The experiments were done in three replicates and the expression levels were normal ized using Phosphoglycerate kinase 1 house keeping gene.@ Statistical analyses The difference between BMP4 and vehicle treated sam ples in cell proliferation and area analysis was evaluated using the Mann Whitney test with GraphPad Prism 4,A P value of less than 0. 05 was considered significant. Results BMP4 inhibits the growth of MCF 10A cells in both 2D and 3D cell culture We began the study using an immortalized breast epithelial cell line MCF 10A, which is widely used in 3D cultures.

However, since no previous data existed, we first tested the effects of BMP4 on these cells in standard 2D culture. Similar to breast cancer cell lines,BMP4 decreased the proliferation of the MCF 10A cells as deter mined by cell counting and alamarBlue,A highly significant decrease in cell number was evident at day 3 and day 6,In 3D assays, both biological and synthetic materials were used.@ In Matrigel, MCF 10A cells formed round acini like struc tures with correct apicobasal polarity of the acini, as illus trated by the basal localization of 6 integrin,In contrast, MCF 10A cells grown in PEG gel demonstrated a disordered structure with no obvious lumen formation and no basal localization of 6 integrin,When MCF 10A cells in Matrigel were treated with BMP4,there was no change in the acinar morphology but proliferation of the cells was reduced,The proliferation rate was decreased by 41% at day 14 in BMP4 treated cells as c