Cted in triplicates on 3 sets of plates with 150 nM siRNA (supplied by the high throughput screening facility at the Center for Genomic Regulation) and Dharmafect four (Dharmacon, Lafayette, CO) according to manufacturer’s guidelines. The cells grown around the plates were handled until d9 as described above. On d9, cells have been treated with two M PMA for two hr at 37 and processed for MUC5AC secretion as described inside the Mucin secretion assay. The Mucin secretion assay was automated and performed on the Caliper LS staccato workstation. Every plate was normalized by the B-score strategy (Brideau et al., 2003) and positive hits had been selected above B-score 1.five and under B-Score -1.5. The hits have been classified applying the ranking solution method (Breitling et al., 2004) using the triplicates. The information was analyzed and automated by a script written with the statistical toolbox from Matlab (Mathwork). The validation screen was performed precisely as described for the screen process. The ontarget PLUS siRNAs had been obtained from 4311-88-0 Cancer Dharmacon (Lafayette, CO). Each of the plates were normalized platewise by:z-score = ( xi average(xn) ) /SD( xn ),xn = total population and xi = sample. Good hits had been chosen 2 SD above and under mock treated samples.Immunofluorescence analysisUndifferentiated and differentiated N2 cells were grown on coverslips. For the visualization of intracellular MUC5AC cells had been fixed with 4 PFA/PBS for 30 min at RT. Cells had been washed with PBS and permeabilized for 20 min with 0.two 1211441-98-3 manufacturer Triton X-100 in four BSA/PBS. The anti-MUC5AC antibody was added for the cells at 1:1000 in 4 BSA/PBS for 1 hr. Cells were washed in PBS and incubated with a donkey anti-mouse Alexa 488 coupled antibody (Invitrogen), diluted at 1:1000 in 4 BSA/PBS, and DAPI. Cells were washed in PBS and mounted in FluorSave Reagent (Calbiochem, Billerica, MA). For the detection of secreted MUC5AC, differentiated N2 cells have been treated with two PMA for two hr at 37 . The secreted MUC5AC was fixed around the cells by adding PFA to the cells at a final concentration of four for 30 min at RT. The cells were then processed for immunofluorescence analysis (as described before) without the permeabilization step with Triton X-100. For the removal of secreted MUC5AC, cells had been incubated for 2 hr with two PMA at 37 . The cells had been then placed on ice and washed 2with ice cold PBS. Subsequently, cells were incubated in 1 mM DTT/0.05 TrypsinEDTA 1(Invitrogen)/PBS for 10 min at 4 , following four washes in ice-cold PBS and two washes in 4 BSA/PBS. The cells had been then fixed in four PFA/PBS for 30 min at room temperature, permeabilized with 0.2 Triton X-100 in 4 BSA/PBS and processed for immunofluorescence as described just before. Cells were imaged using a confocal microscope (SP5; Leica) applying the 63Plan Apo NA 1.4 objective. For detection, the following laser lines have been applied: DAPI, 405 nm; and Alexa Fluor 488, 488 nm; Alexa Fluor 568, 561 nm. Pictures have been acquired using the Leica computer software and converted to TIFF files in ImageJ (version 1.44o; National Institutes of Well being).Pulse chase experimentDifferentiated N2 cells grown on six-well plates were starved in methionine- and cystine-free DMEM (Invitrogen, Carlsbad, CA) for 20 min at 37 . Cells were labeled with 100 Ci 35 S-methionine for 15 min and chased for 3 hr at 37 in medium supplemented with ten mM L-methionine. Brefeldin A (BFA) Sigma-Aldrich was added at a concentration of 2 /ml for the duration of starvation, pulse and chase. The supernatant was collecte.