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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. 6 — Analysis of HPV modulation of DD response upon SMAD4 silencing. A, B HPV-positive (UMSCC104) and HPV-negative (UMSCC18) HNC cell lines were transfected with specific siRNA against SMAD4 or Luciferase as control. 72 h after transfection cells were lysed and analysed by immunoblotting with the indicated antibodies (A) or total RNAs were isolated for RT-qPCR and reported as means ±SD of fold changes of at least three independent experiments. *, P < 0.05; **, P < 0.01; ****, P < 0.0001 (unpaired t test) (B). C, D HKs were transduced with empty or HPV16 E6/E7 recombinant retroviral vectors. After selection with G418 cells were transfected with specific siRNA against SMAD4 or Luciferase as control. 72 h after transfection cells were lysed and analysed by immunoblotting with the indicated antibodies (C) or total RNAs were isolated for RT-qPCR and reported as means ±SD of fold changes of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (One-way Anova Multicomparison test) (D). E, F UDSCC2 cell line was transfected with specific siRNA against SMAD4 and HPV16 E6/E7 alone or in combination, or Luciferase as control. 72 h after transfection cells were lysed and analysed by immunoblotting with the indicated antibodies (E) or total RNAs were isolated for RT-qPCR and reported as means ±SD of fold changes over siLUC. *, P < 0.05, ns, not significant (One-way Anova Multicomparison test) (F). G UDSCC2 cell line was transfected with specific siRNA against SMAD4 or Luciferase as control. 48 h after transfection cells were treated for 24 h with 10 μM Cisplatin and subsequently lysed and analysed by immunoblotting with the indicated antibodies. H UDSCC2 cell line was transfected with specific siRNA against SMAD4 or Luciferase as control. 24 h after transfection cells were seeded into 96well plates (7000 cells/well) and 24 h later were treated with 10 μM Cisplatin for 24 h. Cell viability was then assessed using CellTiter-Glo® Luminescent Cell Viability Assay and expressed as viability relative to untreated cells (means ±SD of fold changes) of two independent experiments. *, P < 0.05; (One-way Anova Multicomparison test)