The sections were observed and imaged by Leica confocal fluorescence microscope

a 20 ml TaqMan MicroRNA Assay using the RNU44 or dme-miR-7 probe set with TaqMan Universal PCR Master Mix in the 7500 Fast Real-Time PCR system, exactly as described by the manufacturer. The CT analysis for each reaction was performed using the supplied 7500 Software v2.0.5 and miR-7 levels were normalized to RNU44 and relative expression was calculated using the 2-DDCT method. 1235481-90-9 site Western blot analysis Total cell lysates were prepared in RIPA Buffer containing Complete EDTA-free Protease Inhibitor Cocktail and PhosSTOP phosphatase inhibitor. Lysates were solubilized in NuPAGE LDS Sample Buffer with NuPAGE Sample Reducing Agent added to 1X. Lysates were separated by electrophoresis with 20 mg of protein per lane in a NuPAGE 412% Bis-Tris Gel and transferred to an Immobilon-FL 0.45 mm Pore Size Transfer Membrane using a Trans-Blot Semi-Dry Electrophoretic Transfer Cell. Membranes were blocked and all antibody dilutions were performed in 5% BSA in TBST. All washes were performed in TBST. Primary antibodies included a-tubulin 05829, HER2 MS-325, EGFR 4267, PAK1 2602, Src 2110, Phospho-Src Y416 6943, FAK 3285, and FAK Y576/577 3281. Secondary antibodies were Alexa Fluor Conjugated Affinity Purified Anti-Rabbit or Anti-Mouse IgG detected using an Odyssey Infrared Imaging System. Colony formation assay Cells were plated at 1,000 cells per well in a 6-well plate PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19681699 and media was replaced every two days for 12 days total. For trastuzumab treatments, trastuzumab was added to media at 10 mg/ml and media was replaced with fresh control or trastuzumab containing media every two days during the experiment. Colonies were fixed in 100% methanol and stained with crystal violet. Colony number and diameter were calculated using a ColCount Colony Counter and data was analyzed using the supplied statistical software. 4 / 16 MiR-7 Suppresses HER2D16 Oncogenic Activity Cell cycle analysis Cells were synchronized in serum-free MEM for 24 hrs and then cultured in MEM with 10% FBS for an additional 24 hrs. Trypsin treated cells were fixed in 100% ethanol overnight. Fixed cells were stained with Guava Cell Cycle Reagent and cell cycle was analyzed in a Guava Easy Cyte Mini Base System using the supplied statistical software exactly as described previously. Cell migration assay Cell migration was determined using the xCELLigence System with the CIM-Plate 16 and RTCA DP Instrument according to the manufacturer’s instructions. Briefly, 40,000 cells were added to the upper CIM-Plate 16 chamber in media containing 0.2% fetal bovine serum. Media with 10% FBS was added to the lower CIM-Plate 16 chamber and the CIM-Plate 16 was incubated in the RTCA DP Instrument for 48 hrs. Cell migration as a function of real-time changes in electrical impedance was monitored by the xCELLigence System. Cell Index and standard deviation of replicates were calculated using the supplied RTCA Software. Results and Discussion HER2D16 alters expression of multiple miRs involved in breast tumorigenesis Clinical and experimental evidence suggests that wild-type HER2 is a relatively weak breast oncogene when compared to the aggressive therapeutic refractory phenotype associated with breast tumors expressing the constitutively active HER2D16 isoform. MiRs potentially regulate PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19682429 multiple properties of tumorigenesis and therapeutic response. Accordingly, we have recently shown that HER2D16 expression alters expression of miR-15a/16 and miR-342 to promote endocrine resistance of breast tumor cells. To inve