4) and then used to treat cells at the indicated doses. Sorafenib (kindly provided by Bayer HealthCare, Germany) and lapatinib (purchased from GlaxoSmithKline plc) were prepared in dimethyl sulfoxide. Solvent alone was added to the untreated cells as the control
in each experiment. Cell viability was assayed at 24 hours www.selleckchem.com/products/MK-2206.html and 48 hours after treatment; for UV treatment, the assays were performed 24 hours after exposure to 30, 65, or 100 mJ/cm2 of UV-B. Cell viability/survival was determined using WST1 assays (Roche Applied Science, Mannheim, Germany), while cell apoptosis was measured by detection of caspase-3 activation (Caspase 3/7 Assay, Promega) as described.7 The experiments were conducted at least twice in triplicate, and the mean of each dose was used to calculate the half maximal inhibitory concentration. Details regarding antibodies, immunoblotting, and immunohistochemistry, immunofluorescence, and confocal microscopy are provided in the Supporting Information.22 Anti-NPM mouse monoclonal
antibody and anti-BAX rabbit polyclonal antibody were used as primary antibodies and anti-mouse and anti-rabbit antibodies coupled with short complementing DNA strands were used as secondary antibodies. Ligation of the DNA strands to a circularized Crizotinib oligomer in case of direct contact between NPM and BAX and the subsequent rolling circle amplification incorporating labeled nucleotides was performed using the Duolink II kit (Olink Bioscience, Uppsala, Sweden) according to the manufacturer’s instructions. After being washed and counterstained with 4′,6-diamidino-2-phenylindole, the slides were mounted and inspected under a fluorescence microscope. Details regarding subcellular fractionation and co-immunoprecipitation are provided in the Supporting Information. The
tumors and their paratumor liver tissues and related clinical data (all anonymous) from 110 patients with HCC were requested from the Taiwan Liver Cancer Network. Normal liver tissue was obtained from a patient with focal nodule hyperplasia who had undergone tumor resection. The Internal Review Board for Medical Ethics of Chang Gung Memorial Hospital approved the specimen collection procedures, and informed consent was obtained from each subject or subject’s family. All HCC tissues selleck screening library were reviewed, and the most representative areas of embedded tissue samples were carefully selected and sampled for the tissue microarray blocks. Two core samples were selected from different areas of each HCC tissue. The immunohistochemistry (IHC) scores are defined as follows: 0, negative; 1, weakly positive or in <20% of HCC cells; 2, moderately positive or in 20% to 60% of HCC cells; 3, strongly positive or in >60% HCC cells. The IHC scores were determined by two independent observers (S. J. L. and T. C. C.), where there was disagreement, the slides were re-examined and a consensus was reached by the observers. A Fisher’s exact test was used for comparison between variables.