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. 2019 Sep 3;9:12680. doi: 10.1038/s41598-019-49061-9

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Significant increase of endogenous p53 levels in HPV-infected cells in the presence of p53 DBD Nbs. FLAG-tagged p53 DBD Nbs were transiently expressed in HPV-infected cells (HeLa (a) and SiHa (b)) or HPV-negative cells (U2OS (c)). Crude lysates (60 µg (HeLa and SiHa) or 40 µg (U2OS)) were prepared 24 h after transfection and p53 expression levels were evaluated through western blotting. In the negative control, cells were transfected with a FLAG-tagged GFP Nb (C). Quantitative analysis of protein levels was performed via ImageJ. Vinculin was used as loading control. Values are represented as the mean fold difference in p53 levels (±SEM) relative to the p53 levels measured for the negative control in 4 independent experiments. Statistical analysis was performed via a one-way ANOVA, with Dunnett’s multiple comparison test with a p-value < 0.05. (a) p53 levels were significantly increased in HeLa cells in the presence of p53 DBD Nb100 (p < 0,05, 2.8-fold increase of p53 levels), p53 DBD Nb105 (p < 0,01, 3.28-fold increase of p53 levels) and p53 DBD Nb120 (p < 0,001, 5.54-fold increase of p53 levels). (b) p53 levels were significantly increased in SiHa cells in the presence of p53 DBD Nb6 (p < 0.05, 2.12-fold increase of p53 levels), p53 DBD Nb100 (p < 0,05, 1.94-fold increase of p53 levels) and p53 DBD Nb120 (p < 0,001, 2.90-fold increase of p53 levels). (c) The p53 levels were unaffected by the presence of p53 DBD Nbs in U2OS cells. For reasons of clarity and conciseness, blots were cropped to the bands of interest. Full-length blots are depicted in Supplementary Fig. S10.