AT7519 were treated with anisomycin for 20 min

VSVtagged Mps1 mutants were obtained from the latter construct by PCR based site directed mutagenesis. AT7519 Cell culture and transfections. Human BJ Tert foreskin fibroblasts were grown in a 1:4 mixture of Medium 199 DMEM with 15 FCS and penicillin streptomycin. Human U2OS osteosarcoma cells and murine JNK1 2 doubledeficient embryonic fibroblasts were cultured in DMEM 8 FCS penicillin streptomycin. Transfections were performed using the standard calcium phosphate protocol. Cells were arrested in mitosis by treatment with 1 mM taxol or 250 ng ml nocodazole for 18 h, and mitotic cells were collected by shake off. Immunoblots and kinase assays. Immunoblots were performed as described previously. To activate JNK1, cells were treated with anisomycin for 20 min.
To obtain active MPS1, Cyclin B Cdc2 or BubR1, cells were incubated overnight with nocodazole and mitotic cells were isolated by shake off before lysis. In vitro kinase assays were performed as described previously Danoprevir using a standardized kinase buffer in the presence of 0.25 mg ml substrate, 50 mM ATP and 2.5 mCi ATP. Phosphorylated substrates were separated on SDS polyacrylamide gels and analysed on blots by autoradiography. Equal precipitation of the kinases was confirmed by probing the blots with the appropriate antibody. Cell cycle analysis and immunofluorescence. Cell cycle distribution and mitotic indices were determined by combined propidium iodide and p histone H3 staining, as described previously. Kinetochore localization of BubR1, Mad1 and Mps1 was judged by analysis of individual prometaphase cells by confocal microscopy as described previously.
Supplementary information is available at EMBO reports online http: www.emboreports.org. Otitis media is the most common medical problem for which children see a physician. Despite otalgia and temporary hearing loss, a single episode of acute OM is not usually a serious concern. On the other hand, recurrent acute OM and chronic OM have been associated with numerous adverse longterm sequelae, including conductive and sensorineural hearing loss, impaired speech and language development, impaired academic achievement, and irreversible middle ear disease. Hyperplasia of the ME mucosa is an important component of OM, involving substantial cell proliferation and differentiation . Hyperplasia contributes to the deleterious sequelae of OM, including the production of mucous secretions of ME effusions.
Hyperplasia is also involved in fibrosis and other permanent damage that can occur in repeated and or chronic OM. The regulation of mucosal hyperplasia in the ME is therefore of clinical significance. Three major groups of distinctly regulated mitogen activated protein kinase cascades are known to lead to altered gene expression and to tissue proliferation in mammals, including extracellular signal regulated kinase 1 2 , Jun N terminal protein kinase, and p38 MAPK. Recent studies in our laboratory investigated the roles of ERK and p38 in OM. It was found that both ERK and p38 can be activated in OM and that inhibition of either the ERK or the p38 pathway can reduce ME mucosal hyperplasia in vitro. Others have found that p38 inhibition reduces bacterially induced mucin gene expression in human ME epithelial cell lines.