Journal: Journal of Virology
Article Title: Cellular transcription factor TFII-I represses adenovirus gene expression
doi: 10.1128/jvi.00618-25
Figure Lengend Snippet: TFII-I KO does not alter the DDR or IFN response to Ad5 infection. TFII-I KO cells were generated by infecting parental BEC, NBS1-, or HDF cells with a CRISPR-Cas9 and guide RNA containing Lentivirus, followed by clonal selection. TFII-I KO was confirmed via Western blot (A, BEC; C, NBS1-; and G, HDF); α-tubulin or vimentin was measured as loading controls. Two independent TFII-I KO clones were selected for each cell type. ( B ) Parental and TFII-I KO BECs were infected with E4ORF3 - /E4ORF6 - Ad5 at an MOI of 1, and total cellular DNA was isolated at the indicated times post-infection. HAdV genome levels were quantified by qPCR and normalized to endogenous GAPDH levels. The results shown represent the mean values ± SD; n = 3. ( D ) NBS1 - (NBS/vector), NBS1 - ectopically expressing Nbs1 (NBS/Nbs1), and NBS1 - TFII-I KO cells (NBS TFII-I-/- #8 and #9) were infected with E4ORF3 - /E4ORF6 - Ad5 (dl355/inORF3) at an MOI of 1, and total cellular DNA was isolated at the indicated time post-infection. HAdV genome levels were quantified by qPCR and normalized to endogenous GAPDH levels. The results shown represent the mean values ± SD; n = 3. ( E ) Parental and TFII-I KO BECs were pretreated for 24 h with IFNα. IRF7 levels were measured by Western blot; Vimentin was measured as a loading control. ( F ) Parental and TFII-I KO HDFs were pretreated for 24 h with IFN-α or IFN-γ and infected with Ad5-WT at an MOI of 1. Total cellular DNA was isolated at the indicated times post-infection. HAdV genome levels were quantified by qPCR and normalized to endogenous GAPDH levels. The results shown represent the mean values ± SD; n = 3.
Article Snippet: The antibodies used were as follows: rabbit polyclonal TFII-I antibody (Cell Signaling Technologies) (1:1,000 dilution), rabbit polyclonal IRF7 (Cell Signaling Technologies) (1:1,000), mouse monoclonal hexon antibody (MAB0850; Abnova) (1:5,000 dilution), rabbit polyclonal penton antibody (gift from Carl Anderson, Brookhaven National Laboratory) (1:1,000 dilution), rabbit polyclonal IVa2 antibody ( ) (1:1,000 dilution), rabbit polyclonal L1-52/55K antibody ( ) (1:1,000 dilution), rabbit polyclonal V antibody (gift from David Matthews, University of Bristol) (1:1,000 dilution), rabbit polyclonal L4-100K antibody ( ) (1:1,000 dilution), rabbit polyclonal VIII antibody (gift from Ann Tollefson and William Wold, St. Louis University) (1:400 dilution), rabbit polyclonal E1A antibody (SC430; Santa Cruz Biotechnology) (1:500 dilution), rabbit polyclonal DNA binding protein (DBP) antibody (gift from Peter van der Vleit, University Utrecht, Netherlands) (1:1,000 dilution), mouse monoclonal α-Tubulin (Sigma-Millipore #T5168) (1:10,000), rabbit polyclonal GAPDH (Millipore Sigma Aldrich) (1:7,500), and rabbit monoclonal vimentin (NeoMarkers) (1:2,000).
Techniques: Infection, Generated, CRISPR, Selection, Western Blot, Clone Assay, Isolation, Plasmid Preparation, Expressing, Control
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