Recombinant adenovirus encoding for human p27kip1 was a kind gift from Professor Betsy Nabel (NIH, MD, USA). This virus and a control adenovirus expressing destabilized, enhanced green fluorescent protein (dsEGFP) were used as described previously (19). To generate the adenovirus expressing E2F1, the plasmid E2F1 wt-pGex2TK containing the coding sequence for human E2F1 from William Kaelin (20), was purchased from Addgene (Addgene plasmid # 21668). It was amplified using KOD DNA polymerase (Merck-Millipore, UK) to include EcoRI and BamH1 flanking sites and subcloned into the shuttle vector pDC515io from Microbix (ON, Canada). Recombination (Flp/FRT mediated) was performed in 293IQ cells to inhibit transgene protein expression (a gift from Dr. D. Matthews, University of Bristol) (21). For gene silencing, short hair pin (Sh) sequences were predicted using http://cancan.cshl.edu/RNAi_central/RNAi.cgi?type=shRNA. The sequences used to silence KDM6A, 5′CTGCCATTAAATGCTACTTAAATAGTGAAGCCACAGATGTATTTAAGTAGCATTTAATGGCAT3′, KDM6B, 5′CGCCCAGTCTGTGAAACCGAAGTAGTGAAGCCACAGATGTACTTCGGTTTCACAGACTGGGCA3′ and firefly luciferase, 5′CGCCTGAAGTCTCTGATTAATAGTGAAGCCACAGATGTATTAATCAGAGACTTCAGGCGGT3′, as a control, were embedded in the backbone of the primary microRNA-30, as described previously (22) and were synthesized by Eurofins. DNA sequences for modified microRNA-30 were synthesized by Eurofins, cloned into the Nhe1-BamH1 sites of the shuttle vector pDC515 and adenoviruses were generated as described above. Virus stocks were purified by CsCl banding and titrated by plaque assay. Monocytes differentiated for 4 days were infected with Ad-p27kip1 or Rad66 at 108 plaque forming units (pfu)/ml for 24 h. Alternatively, cells were infected with Ad-E2F1 or Ad-dsEGFP at 5 × 107 pfu/mL for 20 h followed by further 18 h treatment in the presence of IFN-γ or IL-4. For gene silencing, cells were infected with shKDM6A, shKDM6B, or shLuciferase (shLUC) viruses at 2 × 108 pfu/ml for 72 h before stimulation with IFN-γ or IL-4 for 6 h.