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Pharmacological inhibitors that target pathways within the neuron may alter the behavior of immune cells and regulatory factors that control the virus within a neuron may be essential for immune function or even the viability of the experimental animal. Superior cervical ganglia neurons from E21 rat embryos were dissociated in trypsin at 37 C for 30 min. Approximately 5000 neurons per well were plated in a 96 well plate coated with rat tail collagen. SCG neurons isolated in this manner provide a relatively pure population of neurons expressing the TrkA receptor and contain few non neuronal cells. The cells were maintained with neurobasal media, glucose, B27 supplement, NGF and glutamine and treated with 5 fluorouracil and aphidicolin to eliminate any dividing cells that contaminate the cultures.

After 6 d, the cells were pretreated with acyclovir, for 20 h, and subsequently infected with HSV 1 _ 1 based upon titer on Vero cells) for 2 h in the presence of ACV to block productive HSV 1 replication. Neurons were maintained in ACV for at least 6 d. After ACV removal, infected neuronal CHIR-258 cultures were exposed to different reactivation stimuli. In an experiment, 22 independently infected wells were analyzed per individual stimulus. Graphs summarize a minimum of 3 separate experiments and error bars indicate the standard error of the mean. RNA was isolated from approximately 30,000 latently infected neurons and analyzed by standard methodologies. The primer sequences are posted in the supplementary section.

Cells were cultured and infected with HSV 1 as described above but plated onto 8 well chamber slides at a density of 104 neurons/chamber. In situ Hybridizationwas performed by adding a mix containing four LAT probes for 5 h at 42 C. LAT specific oligonucleotides were designed against the ~2 kb intron MLN8237 region of HSV 1 strain 17, and were synthesized with a fluorescein tag on the 5 end. All subsequent incubations for immunofluorescence were done at RT. Additional details can be found in the supplement. Lentiviruses expressing shRNAs against rat PDK1 and rat PLC? were generated using a pLVTHM vector that included an mCherry expression cassette. SCG cultures were infected with lentivirus for 12 h prior infection with HSV 1. The efficiency of lentiviral infection as judged by mCherry expression was approximately 90%.

The shRNA sequences are posted in the supplementary section. Regulation of these pathways is mediated DCC-2036 by a series of kinases, phosphatases and various exchange proteins. Mutations occur in many of these pathway elements leading to uncontrolled regulation and aberrant signaling. An overview of the effects of mutations and the activation of these signaling pathways is presented in Figure 1. Deregulated signaling can lead to unrestrained cellular growth and proliferation ultimately resulting in tumor formation or abnormal cellular growth and premature aging. As such, a great deal of research has been aimed to target these mutated proteins to prevent abnormal signaling.

Some cancer cells carrying BRAF mutations are highly sensitive to MEK inhibitors, while cells lacking these BRAF mutations or containing RAS or epidermal growth factor receptor mutations are resistant.