Field situations with a Rotaphor six.0 (Biometra, biometra.com). Nuclei from handle and Brd4 knockdown cells had been isolated by hypotonic lysis and micrococcal nuclease assays performed as described by Carey and Smale22. Flow cytometry U2OS cells have been plated and transiently transfected GFP transgenes or siRNA as indicated, exposed to varying doses of ionising radiation from a 137Cs Gammacell Random Inhibitors targets irradiator supply (Atomic Power of Canada, Ltd.), and harvested at varying times as indicated by fixation with four formaldehyde (cell death measurments) or straight extracted with one hundred ethanol (cell cycle measurements), and processed for flow cytometry using the antibodies listed above. Data had been analyzed applying FlowJo (flowjo.com) software program.Author Manuscript Author Manuscript Author Manuscript Author ManuscriptNature. Author manuscript; obtainable in PMC 2013 December 13.Floyd et al.PageColony formation assaysAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptControl and BRD4 knockdown cells had been exposed towards the indicated doses of IR from a 137Cs supply within a Gammacell irradiator (Atomic Energy of Canada, Ltd.), or left untreated, trypsinized, counted and re-plated making use of serial dilutions. Colonies had been propagated for the 105 cell stage (three days), stained with Wright stain (Sigma) and counted with CellProfiler software or by averaging counts of ten fields from 3 independent observers utilizing a dissection microscope to determine colonies of higher than 15 cells. Constructs, shRNA and siRNA, and transfection Full-length constructs of Brd4-NUT (accession #AY166680.1), Brd4 Isoform A (accession # NM_058243), B (accession #BC035266) and C (accession #NM_014299.2) have been cloned into pEGFP-C1 (Clontech) and pFLAG-CMV2 (Sigma) by PCR. Bromodomain mutations were introduced using quickchange (Stratagene) using PCR primers: 5-AAA TTG TTA CAT CGC CAA CAA GCC TGG AGA TGA CGC AGT CTT AAT GGC AG-3 and 5CTG CCA TTA AGA CTG CGT CAT CTC CAG GCT TGT TGG CGA TGT AAC AAT TT-3. Cells were transfected applying Fugene six (Roche) as outlined by manufacturer’s directions. shRNA directed against Brd4 have been in the TRC library (see Table S1), or created in the mir30-based pMLP vector (kind present of Dr. Michael Hemann, MIT, Cambridge, MA, USA) with primer 5-TGC TGT TGA CAG TGA GCG AAG ACA CA-3 for Brd4. U2OS cell lines stably expressing this shRNA or manage hairpins (ineffective hairpins directed against human sequences of Undesirable and PUMA) had been produced applying puromycin selection at 2 g/mL. STEALTH siRNA against pan-isoform BRD4, SMC2, and handle were purchased from RP 73401 Purity Invitrogen. Custom Brd4 isoform-specific siRNA had been synthesized from Dharmacon applying the sequences: Isoform A specific 5-GGG AGA AAG AGG AGC GUG AUU-3 and Isoform B precise 5-GCA CCA GUG GAG ACU UCG UUU-3. siRNA against SMC2 was from Dharmacon. For siRNA experiments, cells had been transfected with Lipofectamine RNAiMax (Invitrogen) according to manufacturer’s directions. Mass spectrometry Proteins in the Brd4 co-immunoprecipitation were examined immediately after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by staining with Coomassie Blue. Gel bands were excised, de-stained and processed for digestion with trypsin (Promega; 12.five ng/l in 50 mM ammonium bicarbonate, pH eight.9). Peptides had been loaded straight onto a column packed with C18 beads. The column was placed in-line with a tapered electrospray column packed with C18 beads on a Orbitrap XL mass spectrometer (Thermo Scientific). Peptides were eluted u.