Functional consequences of the L2–RNF20 interaction.

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Functional consequences of the L2–RNF20 interaction. Functional consequences of the L2–RNF20 interaction. A, Subnetwork of HPV L2 interacting with RNF20 and RNF40. Green/blue arrows represent the main contribution of MiST/differential mutation scores through network propagation, respectively. MiST scores of virus–human interaction in C33A cells is indicated (0.82, 0.69). B, C, E, Western blot analysis of virus–host interaction by IP using streptavidin-coated beads to bind the Strep-tag. Proteins indicated on the right of each blot from IP and input samples were detected using the antibodies indicated. Bands were cropped from the same original membrane. Distinction between different strains of HPV is made using strain number preceding the respective viral bait (HPV-31 = 31, HPV-16 = 16; C). D, Cartoons depicting the truncation/deletion mutants of HPV-31 L2. Scale is provided in amino acid numbers of the full-length protein at the top. F, Expression level of upregulated EMT genes in response to L2 overexpression (see also Supplementary Table S6). The z-score normalized expression of each gene is indicated by color intensity according to the color scale. G, Transwell invasion assay of Het-1A human esophagus cells overexpressing HPV-31 L2 or GFP. Representative images of the bottom side of the Transwell after invasion of Het-1A cells overexpressing HPV-31 L2 (top) and GFP (bottom). H and I, Quantification of Transwell invasion assays. Individual dots with centerline (mean) and error bar (standard deviation) show the number of invaded cells per image (P value from two-tailed t test) of Het-1A cells overexpressing HPV-31 L2 and GFP (H) and control UD-SCC2 hypopharyngeal cancer cells compared with polyclonal disruptions of RNF20 (I). J, Western blot analysis of UD-SCC2 hypopharyngeal cancer cells subjected to CRISPR/Cas9 gene editing using the indicated guide RNAs. Proteins indicated on the right of each blot were detected using the antibodies indicated. Bands were cropped from the same original membrane. Manon Eckhardt et al. Cancer Discov 2018;8:1474-1489 ©2018 by American Association for Cancer Research