Transfection, Constructs, and RNAi

Cells were transfected 12–18 h following passage at 70–80% confluency. At this time complete cell media containing 15% FBS was removed, and replaced with a transfection mixture containing Targefect F-2 (5 μl/ml) and peptide enhancer (5 μl/ml) (Targeting Systems) in serum-free Dulbecco’s modified eagle’s medium (DMEM; Sigma-Aldrich). siRNA was transfected at 50 nM, whilst cDNA was transfected at 250 ng/ml. The transfection mixture was incubated with the cells at 37°C for 4 h, at which point it was removed and replaced with complete growth media containing serum. Cells were maintained in this media for 24–48 h to allow for protein expression or knockdown. Details of our wild type p65, p65 Arg¹⁷⁴Ala and Arg¹⁷⁴Lys cDNA constructs can be found in our previous publication [3]. In brief, human p65 cDNA (Addgene construct 21966) was inserted into pCDNA3 with an N-terminal FLAG epitope tag (AA: DYKDDDDK) in frame with the start codon and expressed under the control of the CMV promoter. Arg¹⁷⁴ was mutated using the PCR-based GeneArt Mutagenesis System (Life Technologies). We repeated all experiments using two different siRNA sequences for each target to minimize the potential of off-target effects. siRNAs complementary to the PRMT5 coding sequence were sense 5´-GAGGGAGUUCAUUCAGGAAUU-3´, and PRMT5 3´ UTR, sense 5´-GCUCAAGCCACCAAUCUAUUU-3´. siRNAs targeting the NF-κB p65 3´ UTR were sense 5´-GGAUUCAUUACAGCUUAAUUU-3´ and sense 5´-GCUCUUUCUACUCUGAACUUU-3´. All siRNAs were designed using the Whitehead Institute siRNA design tool (http://sirna.wi.mit.edu/) and synthesized by Ambion containing the Silencer Select modifications [3]. Nontargeting Silencer Select siRNA was used as a control (Ambion).