Targeting JNK1 may provide a novel therapeutic approach for treating pneumonia

death after loss of contact with the extracellular matrix, have been found to correlate with transformation, tumorigenic activity, tumor progression, and metastasis. Molecules that confer these properties on cancer cells have remained to be definitively identified, however. We have now compared the gene expression profiles of AX and AXT cells and have identified the gene for Imp3 as being MedChemExpress CAL 101 highly overexpressed in AXT cells. We further found that Imp3 plays a key role in the anchorageindependent growth and anoikis resistance in vitro as well as in their tumorigenicity in vivo. Our findings thus indicate that Imp3 is a potential target for therapeutic control of the aggressiveness of osteosarcoma. Imp3 Activates Osteosarcoma Tumorigenesis In Vivo Materials and Methods Cell Culture Mouse osteosarcoma AX and AXT cells were established as previously described and were cultured in DMEM High Glucose supplemented with 10% FBS and antibiotic-antimycotic. In the experiments of inhibition of DNA methyltransferase and/or histone deacetylase, AX cells were treated with 5-AZA-29DEOXYCYTIDINE , TRICHOSTATIN A , Valproic acid or SAHA at the indicated concentration for one day. Cells were collected and subjected to RT and realtime PCR analysis. Flow Cytometry Cells were stained with FITC-conjugated annexin V and propidium iodide with use of apoptosis detection kit and were analyzed with FACS Calibur. Cell Proliferation Assay Cells were transferred to 96-well tissue culture plates or 96-well ultra low-adherence plates and were cultured in DMEM supplemented with 10% FBS and in the absence or presence of mouse Igf2 as indicated. Cell proliferation was assayed in triplicate with the use of a Cell Titer Glo assay kit. Quantitative data are expressed relative to the value for time 0 and are means 6 SD for three independent experiments. RT and Real-time PCR Analysis Total RNA was extracted from cells or tumors with the use of RNeasy Mini Spin columns and was subjected to RT with a Prime Script RT-PCR kit. Real-time PCR analysis was performed with SYBR Premix Ex TaqII and Thermal Cycler Dice. The sequences of primers are shown in Immunoblot Analysis Cells were lysed with Laemmli sample buffer and subjected to immunoblot analysis according to standard procedures. Primary antibodies included those to IMP3, aTubulin, rpS6, and Ago2. Tumor Xenograft Model Immunostaining Immunohistochemical analysis was performed according to standard methods. Deparaffinized sections were stained with rabbit polyclonal antibodies to GFP-FL or IMP3. Immune complexes were detected with Histofine and Simple Stain kit. For immunofluorescence analysis, cells were fixed with acetone and stained with primary antibodies and Alexa546-conjugated secondary antibodies. Nuclei were stained with TOTO3. Samples were observed with LSM510 confocal microscope and analyzed with LSM image browser. Single-cell suspensions were prepared in 100 ml of PBS and were injected subcutaneously or intraperitoneally into 8-week-old syngeneic female C57BL/6 mice. The weight of subcutaneous tumors was measured after 28 days unless indicated otherwise. Polysome Analysis AXT cells were cultured in 10-cm dishes, washed with ice-cold PBS, and lysed in a solution containing 20 mM HepesKOH, 150 mM NaCl, 2.5 mM MgCl2, 0.1% NP-40, 1 mM DTT and protease inhibitors. The lysate was subjected to centrifugation at 15,0006g for 10 min at 4uC, and the resulting supernatant was applied to 530% sucrose density gradient prepared in th