Animal experiments The animal experiments were performed as described previousl

1% ethanol was used as a control. Immunofluorescence Cells were plated on 10 mm diameter cover slides in 24 well plates. After 48 h, the cells were fixed for 10 min in phosphate buffered saline containing ABT-888 溶解度 4% paraformaldehyde. The cells were then permeabilized in PBS containing 0. 3% Triton X 100 for 10 min. The primary antibodies were diluted in PBS containing 3% FCS and added to the permeabilized cells, which were incubated over night at 4 C. Dye conjugated secondary antibodies were incubated 1 h at room temperature. After mounting in Vectashield mounting medium with DAPI, images were obtained using an Imager. Z1 ApoTome AxioCam epifluorescence microscope and proc essed with Axio Vision Software. RT PCR assays 2. 5 × 105 cells were cultured in 6 well plates and treated as specified.<br><br> Total RNA was extracted, at least in tripli cate, using the Trizol reagent according to the manufacturers instructions. cDNA was generated Afatinib 臨床試験 by MMLV Reverse transcriptase using random hexamers. Quantitative real time RT PCR was performed using the iQ SybrGreen supermix and a Bio Rad MyiQ apparatus. The primers used for the cDNA amplifications in the quantitative RT PCR ex periments are described in Table 1. GAPDH and RNA 18S were used as housekeeping genes to normalize the expres sion levels of the genes of interest. GAPDH was found to be appropriate for normalisation in cell lines because its expression was not affected by treatments and remained stable in control and COUP clones. For tissues, we first verified the choice of the reference gene as an internal con trol and its suitability in our study.<br><br> Four housekeeping genes were tested. The stability of these genes across different tissues and tumor grades was assessed using geNorm algorithm. This software has listed HPRT1 as the best gene but HPRT1 is expressed at very low level in normal tissues and tumors, making it quite difficult to accurately quantify AG-1478 構造 and not enough useful as an internal reference in our study. The second best gene, established by the software in the list, was the 18S RNA. This RNA is expressed similarly at relatively high levels in all tumors and made ideal posi tive control for our study. Thus, we have chosen 18S for normalization. Melting curves and PCR efficiency analyses were per formed to confirm correct amplification. Each experi ment was performed at least three times.<br><br> Results were expressed according to the comparative Ct method for relative quantification of gene expression. For each sample, the difference was calculated between Ct values obtained for target and reference amplicons. Comparative ddCt was then determined using as a refer ence the dCT calculated for the vehicle control sample, and absolute values for comparative expres sion level were determined as equal to 2 ddCt. Protein extraction Western blotting Total proteins were extracted in RIPA buffer with an anti protease mixture and quantified using the Bio Rad DC protein assay kit. The proteins were diluted in Laemmli buffer and dena tured at 95 C, 30 ug of denatured proteins were separated on SDS polyacrylamide gels, transferred to polyvinylidene difluoride membranes, and probed with specific antibodies.