Treatment with P7170 resulted in a significant growth inhibition of xenograft

RAD001 in vitro experiments cells were pre treated for 30 minutes with 20 nM RAD001 followed by 100 pM E2 or DMSO. miRNA was reversetranscribed using the SABiosciences RT2 miRNA first strand kit and qPCR was per formed using SABiosciences SYBR green, miR 155 pri mer, U6 primer, and SA Bioscience RT2 cancer miRNA array plate were purchased from Qiagen. small molecule Data was analyzed by comparing rela tive target gene expression to B actin for mRNA and U6 for miRNA. Relative gene expression was analyzed using 2 Ct method. Transfection of Cell Lines miR 155 and vector plasmid were generated as previ ously described. MCF 7 cells were transfected with pre mir 155 or vector plasmid using Lipofectamine 2000 at 1ugul OPTI MEM as per manufacturers protocol. Parental MCF 7 cells were grown in a 100 mm dish.<br><br> 5ug pre mir 155 or vector plasmid was added to 100 ul serum free opti MEM then 15 ul Lipofectamine was added. Following 30 minutes opti MEM containing plasmid was added to MCF 7 cells. The following day cells were treated with 300 ngml puro mycin. Cells were maintained in 10% DMEM and treated with 300 ngml puromycin every two days for 2 weeks. Colonies were pooled and verification Lenalidomide 分子量 of mature miR 155 overexpression was confirmed using qPCR for mature miR 155. Stable pools were maintained in 10% DMEM as described above. For generation of miR 155 sponge, miR 155 sponge sequence was taken from pMSCV puro GFP miR155SPONGE as previously described and inserted downstream from the RFP sequence in the TRIPz RFP vector backbone.<br><br> Sponge was transfected through lenti viral transfection as previously described and retro virus packing was performed following the manufacturers instructions. Crystal Violet Assay MCF 7 vector and MCF オーダー LY2603618 7 miR 155 cells grown in 5% phenol free DMEM for 24 hours and then plated in 48 well plates for 24 hours prior to a one time treatment with 1 nM E2 or DMSO. After 72 hours cells were washed once with PBS and fixed and stained using 0. 1% Crystal Violet for 10 minutes. Cells were washed with water and lysed with 1% SDS. Gene5 plate reader was used to read absorb ance at wavelength 630. Each cell line was normalized to its respective DMSO treated group. Western blot analysis MCF 7 vector andmiR 155 cells grown 10% FBS DMEM supplemented. Cells were washed with PBS and lysed with M Per lysis buffer supplemented with 1% pro tease inhibitor and 1% phosphatase inhibitors.<br><br> Supernatant containing protein extracts was obtained through centrifugation at 12,000 RPM for ten minutes at 4 degrees Celsius. Protein extracted per sample was determined by absorbance at 260 and 280 nm using the NanoDrop ND 1000. Proteins were heat denatured and 40ug of protein were loaded per lane on Bis Tris nuPAGE gel. Protein transfer to nitrocellulose through iBlot and iBlot transfer stacks as per manufacturers protocol. Nonspecific binding was blocked by incubation in 5% BSA in 1% TBS T for 1 hour. Over night incubation of membrane with primary antibody for total mTOR, p mTOR, Rictor, Raptor, TSC1, total p70s6kinase, p 4E BP1, AKT, p p70s6kinase, p S6 ribosomal protein, p eIF4B, p eEF2K diluted 1 1,000 and PgR and ER diluted 1 250 at 4 degree Celsius followed by three fifteen minute washes in 1% TBS T.