Loss of IKK subunits limits NF-?B signaling in reovirus infected cells

Andrew J. McNamara; Pranav Danthi

Infections. Confluent monolayers of ATCC L929 or HEK293 cells were adsorbed with 325 either PBS or reovirus at the indicated MOI at room temperature for 1 h, followed by 326 incubation with medium at 37°C for the indicated time interval. All inhibitors were added 327 to cells in medium after the 1 h adsorption period. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/843680 doi: bioRxiv preprint featureCounts tool from subread package (45). The differential expression analysis was 342 performed using DESeq2 version 1.12.3 (46). 343 iRegulon, a plugin to cytoscape version 3.7.1 was used to predict transcription 344 factor activity based on a differentially expressed gene set (18) The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/843680 doi: bioRxiv preprint mouse Ig (for PSTAIR, RIP1, and FLAG) in blocking buffer. Following three washes, 387 membranes were scanned and quantified using an Odyssey infrared Imager (LI-COR). 388 . CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/843680 doi: bioRxiv preprint The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. Fig. 1A following vgRNA transfection of mock infected and T3A infected cells is shown. Black dots denote genes that are not expressed significantly differently in the two treatments. Red dots represent genes that are expressed to a significantly lower extent in T3A infected cells transfected with vgRNA. iRegulon analyses of both sets of genes is also shown. Following incubation at 37°C for 24 h, cells were treated with 10 ng/ml TNFα and incubated for 1 h. Nuclear extracts were immunoblotted using antiserum specific for p65 or PSTAIR. (C) ATCC L929 cells were adsorbed with PBS (mock) or 10 PFU/cell of T3A in the presence of 0 or 200 µM ribavirin. Following incubation at 37°C for 24 h, cells were treated with 10 ng/ml TNFα and incubated for 1 h. RNA was extracted from cells and IκBα gene expression was measured using RT-qPCR. Gene expression in mock infected cells treated with TNFα was set to 100%. Gene expression of each replicate, the mean value and SD are shown. *, P < 0.05 by Student's t test in comparison to mock infected cells treated with TNFα. NS, not significant in comparison to mock infected cells treated with TNFα.