In vitro transformation assays Anchorage independent growth was assessed by seeding the cells on soft agar and counting the colonies after 14 days using an Tofacitinib Citrate structure inverted microscope and 0. 005% crystal violet for staining. Cell mi gration and invasion was assessed in Boyden chambers using 8um pore inserts, with or without matrigel coating according to the manufacturers instructions. Inhibitors,Modulators,Libraries Animal studies NOD SCID and NSG mice were purchased from The Jackson Laboratory, and cared for in strict accordance with an approved Johns Hopkins ACUC protocol. Intraductal transplantation was performed as described previously. Briefly, 105 cells were injected in the mammary ducts of immunodeficient female NSG mice as 2 uL of single cell suspension in PBS with 0. 1% trypan blue, using a Hamilton syringe with a blunt ended 12 inch 30 gauge needle.
At the indicated times, mice were euthanized, and the mammary fat pads harvested and fixed in 10% neutral buffer formalin. For xenografts, CSE treated cells were subcutaneously injected as a 50 ul single cell suspension in a 1 1 solution of media and BD Matrigel Matrix. At the indicated times, the mice were euthanized, and fixed by perfusion with PBS Inhibitors,Modulators,Libraries followed by 10% neutral buffer formalin for necropsy. Fe male mice receiving MCF7 cells were implanted with bees wax pellets containing 20 ug of estrogen one day before injection. Paraffin embedded sections were analyzed by standard H E staining, and by immunohistochemistry using a monoclonal antibody for human cytokeratin 18, and the Mouse on Mouse Fluorescein Kit.
Flow cytometry Fluorescence activated cell sorting was performed on a BD Bioscience SLRII instrument. Cells were labeled using the ALDEFLUOR kit, or the antibody conjugates listed in the Additional file 3. Analysis of gene expression and methylation Microarray based Inhibitors,Modulators,Libraries gene expression and methylation ana lysis were performed at the microarray core of the SKCCC using the Agilent Human 44K Inhibitors,Modulators,Libraries expression array and the Infinium Methylation27 Array as previously described. The data is depos ited in the GEO database under accession number GSE42668. Quantitative Real Time PCR analysis was performed using a 7500 Real Time PCR System, the High Capacity cDNA Reverse Transcription Kit, TaqMan Gene Expression Master Mix, and the TaqMan Gene Expression Assays listed in the Additional file 3.
Western blot analysis was performed as previously described using the antibodies listed in the Additional file 3. Background PDAC is the fourth leading cause of cancer deaths in the United States and has the worst prognosis of all solid tumors. This year alone it is Inhibitors,Modulators,Libraries estimated that 38,460 of the 45,220 patients diagnosed with pancreatic cancer in the United States http://www.selleckchem.com/products/PF-2341066.html will succumb to the disease. Despite advancements in the understanding of the genetics of this disease and the use of combined chemotherapy and targeted biological agents, the management of this lethal malignancy remains one of the greatest onco logical challenges.