These data identify the JNK1 pathway as an important target in understanding lung immunity

d three times in ice-cold PBS. Cells were resuspended in ice-cold PBS to a final concentration of 206106 cells/mL. Freshly prepared, 10 mM sulfo-NHS-SS-biotin was added to the cell mixture and the mixture was incubated at room temperature for 30 minutes with shaking. 50 mM Tris was added to deactivate the biotinylation reaction, and the cell pellet was subsequently washed twice with PBS. The final PBS wash was removed completely from the cell pellet, and RIPA buffer containing PI was applied to the cell pellet. The lysis reaction was incubated on ice for one hour. The lysate was cleared by centrifuging at 12,000 RPM for 10 minutes, after which the supernatant was removed and stored at 20uC. A BCA assay was performed on the biotinylated lysate as described previously; approximately 1.5 mg of protein was obtained. Avidin agarose was applied to a microcentrifuge tube and the resin was settled by centrifuging 1 minute at 2000 RPM. The resin was washed three times with RIPA buffer. The lysate was applied to the resin and the mixture was incubated on a tube rotator at 4uC overnight. The resin was settled by centrifuging 1 minute at 2000 RPM and the unbound lysate was removed. Unbound lysate was mixed with Laemmli Sample Buffer containing 5% bME. The resin was washed three times with RIPA containing PI. Laemmli Treating Cultures with HAase Inhibitor A solution of 125 mM DSC was prepared in 40% DMSO and filtered in a Steriflip filter to sterilize. DSC was added to 800 ml or 2.4 mL T-medium in 24 or 6 well plates, respectively, to a final concentration of 500 mM DSC, the IC-50 value of this inhibitor. An equal amount of 40% DMSO was used as a vehicle control. Medium was Prostate Cancer Invadopodia in 3D HA Hydrogels changed every three days and fresh DSC or DMSO solution was applied as described. of invadopodia formation and merging clusters are independent of cell division. Invadopodia and cluster formation in a PCa progression model. Next, we evaluated the parameters of invadopodia Trypan Blue Exclusion The toxicity of DSC on PCa cells was determined using a trypan blue exclusion assay. 600,000 cells were plated into 6 well plates with T medium, 5% FBS, 1% PS. After 24 hours of culture, the cultures were treated with DSC or vehicle control as described previously. At three and six day timepoints, medium was aspirated and cells were released with trypsin. Cells were pelleted, then resuspended in 500 uL T medium. 50 uL of a 0.4% solution of trypan blue in buffered isotonic salt solution was added to each sample. The total number of white and blue cells was determined by counting on a hemocytometer. The percentage of live cells was determined from this data. Statistical Analysis Error bars on all figures display standard error of the mean. Significance was determined using Student’s two sample two-tailed t-tests with p,0.05 considered significant. Results Invadopodia and Cluster Formation in 3D HA Hydrogels Invadopodia and cluster formation response to motogenic factors. In initial experiments, we evaluated C4-2 number and merging cluster percentage in the HA hydrogel for PCa cells representing increasing disease progression. For this study we compared highly aggressive, metastatic C4-2 cells to their less aggressive LNCaP parent cell line. Similar DM 1 metabolic activities and cell counts were observed when comparing LNCaP and C4-2 cultures at three and six day timepoints. We hypothesized that the differences in invadopodia number and/or merging clus