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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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Evaluation of p53 levels in HPV-infected cells receiving a co-treatment with p53 DBD Nbs and the HDACi SAHA. HeLa (a) and SiHa (b) cells transfected with FLAG-tagged p53 DBD Nbs received (or did not receive) an additional treatment with 10 µM SAHA for a duration of 20 h. Cells transiently expressing a FLAG-tagged GFP Nb were implemented as negative control (C). Crude lysates (60 µg) were prepared and p53 levels were analysed through western blotting. Vinculin was used as a loading control and quantitative analysis of protein levels was accomplished by means of ImageJ. Values are represented as mean AUC (p53/vinculin) (±SEM) of 3 independent experiments. Statistical analysis was performed using a two-way ANOVA with a Bonferroni post-test. (a) p53 DBD Nbs were responsible for a stabilisation of p53 in HeLa cells, where significant higher p53 levels were observed for p53 DBD Nb120 compared to the negative control (p < 0.01). SAHA treatment of the control cells resulted in a 4.18-fold increase in p53 levels (C/− vs C/+). Nevertheless, the p53 DBD Nbs had a more substantial impact on p53 stability, irrespective of whether or not the cells were co-treated with SAHA. The stabilising effect of p53 DBD Nb105 was slightly enhanced in the presence of SAHA, represented by a 1.36-fold increase in p53 levels. By contrast, SAHA treatment of HeLa cells expressing p53 DBD Nb120 resulted in a 1.66-fold reduction of p53 levels. (b) Expression of p53 DBD Nb120 in SiHa cells caused a significant increase of p53 levels compared to the negative control (p < 0,05). Curiously, SAHA treatment had no impact on the stability of p53, neither in the control condition nor in SiHa cells expressing p53 DBD Nbs. Even more, treatment with SAHA rather promoted a reduction of p53 levels and counteracted the impact of the p53 DBD Nbs. SAHA treatment caused a 7.17-fold or a 4.23-fold reduction of p53 levels in cells expressing p53 DBD Nb105 or p53 DBD Nb120, respectively. Blots were cropped to the bands of interest. Full-length blots are depicted in Supplementary Fig. S13.