As these represent the maximal concentrations that did not compromise N9 cell viability (Additional file

As these represent the maximal concentrations that did not compromise N9 cell viability (Additional file 1: Figure S6). For experiments to evaluate the effect of BET inhibitors on lipopolysaccharide (LPS)-stimulated phenotypes of activated microglia (N9 or primary PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27486068 cells), cells were pre-treated with JQ1, Olinone, or RVX208 for 12 h and then stimulated with LPS (1 g/mL, Sigma-Aldrich, St. Louis, MO, USA) for 2 h followed by various assays as described below in detail.N9 microglia cell PD-148515 custom synthesis Proliferation assay (BrdU)temperature. The upper chamber of the inserts was swabbed again and rinsed twice with PBS. After airdrying the inserts for 30 min, the polyester membranes were harvested using a scalpel and mounted on glass slides using 90 glycerol. Images were then taken to quantify cells that migrated across the membrane from the upper chamber to the lower surface.siRNA knockdown of BET proteinsTo study the effect of BET inhibitors on the proliferation of N9 microglial cells, we used a Cell Proliferation BrdU ELISA (colorimetric) Kit (Roche Applied Science, Indianapolis, IN) following manufacturer instructions, as described in our previous study [22] with minor modifications. Briefly, N9 cells were seeded in 96-well plates at a density of 4000 cells per well with a final volume of 200 l, in DMEM containing 0.5 FBS. Cells were pretreated with 0.5 M JQ1, 30 M Olinone, 30 M RVX208, or an equal volume of vehicle control (DMSO) for 12 h prior to LPS stimulation (final 1 g/ml). After LPS treatment for 2 h, cells were labeled with BrdU in DMEM containing 10 FBS for a 2-h incubation at 37 ?C, and then fixed with a FixDenat solution for 30 min, followed by a 90-min incubation at room temperature with an anti-BrdU-POD antibody (1:100 dilution). After washing with PBS three times, substrate was added. Plates were incubated at room temperature for 30 min, and colorimetric signals were measured on a FlexStation 3 Benchtop Multi-Mode Microplate Reader (Molecular Devices, Sunnyvale, CA) at 370 nm with a reference wavelength of 492 nm.N9 microglia cell migration assay (Transwell)Assay was performed according to our previously reported method [22]. Briefly, N9 cells were seeded at a density of 20,000/well in the upper chamber of Transwell Permeable Supports (or Inserts) (8 m pore size, Corning, NY) placed in 24-well plates. Cells were preincubated with 0.5 M JQ1, 30 M Olinone, and 30 M RVX208 or vehicle (DMSO) for 12 h prior to LPS stimulation (final 1 g/ml). Inserts were harvested at 24-h post stimulation and fixed in 70 ethanol at -20 for 30 min. Pre-moistened cotton swabs were used to gently scrape remaining cells in the upper chamber of inserts, followed by staining the cells on the lower surface of the insert in hematoxylin solution for 30 min at roomKnockdown was performed as described in our previous report [22]. Lentiviruses for expression of scrambled or mouse BET-specific siRNAs were packaged using a three-plasmid expression system including piLentisiRNA-GFP, psPAX2, and pMD2.G (Addgene, Cambridge, MA). The piLenti-siRNA-GFP vectors for expression of a scrambled siRNA or siRNAs specific for mouse BET2 or BET4 were purchased from Applied Biological Materials Inc. (Canada). For BET2 knockdown, two siRNAs were used as a mixture: 5-CCACAATGGCTTCTGTACCAGCTTTACAA-3 5-CCACAATGGCTTCTGTACCAGCTTTACAA-3 For BET4 knockdown, four siRNAs were used as a mixture: 5-GTGGATGCCGTCAAGCTGAACCTCCCTGA-3 5-GGACTTCAACACTATGTTTACAAATTGTT-3 5-GGAGATGACATCGTCTTAATGGCAGAAGC-.