In conclusion, these findings reinforce the role of IFI16 as a mediator of the immunomodulatory and proinflammatory activities of IFN that regulate the early defence mechanisms against infections. HUVEC cultured in endothelial growth medium (EGM-2, Lonza, Milan) containing 2% fetal bovine serum, human recombinant vascular endothelial growth factor, basic fibroblast growth factor, human epidermal growth factor, IGF-1, hydrocortisone, ascorbic acid, heparin, gentamycin and amphotericin B (1 μg/mL each) were seeded into 60 mm culture dishes coated with 0.2% gelatin. Experiments were performed with cells between passages 2 and 6. Human

embryo kidney 293 cells (Microbix Biosystems) were cultured in minimum selleck inhibitor Eagle’s medium (Invitrogen) supplemented with 10% fetal bovine serum (Sigma, Milan, Italy), 2 mM glutamine, 100 units of penicillin per milliliter and 100 μg/mL of streptomycin

sulfate. Adenovirus-derived vectors expressing either IFI16 or LacZ were generated as described previously 9. Briefly, the LY294002 mouse pAC-CMV IFI16 containing the human IFI16 cDNA linked to a FLAG tag at the N-terminus was cotransfected together with pJM17 into human embryonic kidney 293 cells. After several rounds of plaque purification, the AdVIFI16 was amplified on 293 cell monolayers and purified from cell lysates by banding twice on CsCl gradients. Recombinant AdVIFI16 Clomifene was tested for IFI16 expression by Western blotting using an anti-FLAG Ab (Sigma). For cell transduction, preconfluent HUVEC were washed once with PBS and incubated with either AdVIFI16 or AdVLacZ (used as a control) at a MOI of 300 in EGM-2. After 60 min at 37°C, the virus was washed off

and fresh medium added. Cells were cultured for 36 h before use in the experiments. RT-PCR analysis was performed on an Mx 3000 PTM (Stratagene) using the SYBR Green I dye (Fermentas) as a nonspecific PCR product fluorescence label. Total cellular RNA was isolated using the Nucleospin Extract RNA II (Macherey Nagel). RNA (1 μg) was then retrotranscribed at 42°C for 60 min in PCR buffer (1.5 mM MgCl2) containing 5 μM random primers, 0.5 mM dNTP and 100 units of RevertAid H Minus M-MuLV Reverse Transcriptase in a final volume of 20 μL. cDNA (1 μL), or water as control, were amplified in duplicate by RT-PCR using the Brilliant SYBR Green QPCR master mix (Fermentas) in a final volume of 25 μL. Primer sequences are summarized in Table 2. The Ct values for each gene were normalized to the Ct values for β-actin using the Ct equation. The level of target RNA, normalized to the endogenous reference and relative to the mock infected and untreated cells, was calculated by the comparative Ct method using the 2−δδCt equation. For transfection experiments, HUVEC grown to subconfluence were detached and transfected with 0.