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. 2022 Feb 10;41:59. doi: 10.1186/s13046-022-02258-9

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© The Author(s) 2022

Open AccessThis 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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 4 — Analysis of βTRCP1-dependent SMAD4 degradation in HPV-negative and HPV-positive HNC cell lines. A Total RNAs from HNC cell lines were isolated for RT-qPCR. βTRCP1 expression was normalized to RpP0 and shown as Box plot of means ± SD and individual values of at least three independent experiments. *, P < 0.05 (unpaired t test). B Normalized RNA-seq data were extracted from TCGA database and divided in HPV+ and HPV- HNC cohorts. Numbers in brackets refer to the number of samples included in each analysis. ****, P < 0.0001 (unpaired t test). C HNC cell lines were lysed and analysed by immunoblotting with the indicated antibodies. D Box plot showing means ± SD and individual values of Optical density analysis of the expression of βTRCP1 normalised to loading control (GAPDH) from three independent western blot experiments in HNC cell lines. E pcDNA3-βTRCP1-flag plasmid was transfected into HPV-positive (UPCISCC154) and HPV-negative (UMSCC18) HNC cell lines. One day after transfection cell were treated with 5 μM MG132 overnight. Two days after transfection cells were harvested and whole cells extracts were subjected to anti-Flag immunoprecipitation (IP). Immunoblotting with the indicated antibodies was then performed and whole cell extracts were used as input controls. Graph shows Optical density analysis of SMAD4 co-immunoprecipitated normalised to βTRCP1-flag immunoprecipitated. F HPV-positive (UPCISCC154) and HPV-negative (UMSCC18) HNC cell lines were transfected with specific siRNA against βTRCP1 or Luciferase. Two days after transfection cell were treated with 5 μM MG132 overnight. 72 h after transfection cells were harvested and whole cells extracts were subjected to anti-SMAD4 immunoprecipitation (IP). Immunoblotting with the indicated antibodies was then performed and whole cell extracts were used as input controls