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. 2021 Oct 25;12(11):997. doi: 10.1038/s41419-021-04301-7

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© The Author(s) 2021

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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Fig. 5 — a Autophagic flux assay using WSU12 cells treated with 5 μM YOK1104 (12 h) and/or 500 nM bafilomycin A1 (6 h) with or without irradiation at 6 Gy. b Quantification of LC3-II band intensity of a. Error bars represent means ± SD from three independent experiments (***p < 0.001, ns non-significant). c Autophagic flux assay using UM19 cells treated with 5 μM YOK1104 (12 h) and/or 500 nM bafilomycin A1 (6 h) with or without irradiation at 6 Gy. d Quantification of LC3-II band intensity of c. Error bars represent means ± SD from three independent experiments (**p < 0.01, ***p < 0.001, ns non-significant). e Immunoblot analysis of caspase-8 of WSU12 cells in the presence or absence of 5 μM YOK1104 with or without irradiation at 6 Gy. f Caspase-3/7 (DEVDase)-like activity was measured in WSU12 cells treated with 5 μM YOK1104 and 6 Gy XRT in the absence or presence of caspase-8 inhibitor (20 μM, 25 h). ***p < 0.001. g Immunoblot analyses of ubiquitinated proteins and caspase-8 using total WSU12 cell lysates (left panel) or caspase-8 immunoprecipitates (right panel) after treatment with 5 μM YOK1104 with or without irradiation at 6 Gy. h Immunostaining of caspase-8 (green) and p62 (red) in WSU12 HPV(-) HNSCC treated with vehicle control (DMSO) or 5 μM YOK1104 with or without irradiation at 6 Gy. Scale bar: 10 μm. i Immunostaining of caspase-8 (green) and K63-linked ubiquitin (red) in UM19 HPV(-) HNSCC treated with vehicle control (DMSO) or 5 μM YOK1104 with or without irradiation at 6 Gy. Scale bar: 10 μm. j Analysis of subcellular localization of caspase-8 (green) using a single focal plane at the middle of the nucleus (DAPI, blue). k Immunostaining of caspase-8 (green) in control and p62 KD WSU12 HPV(-) HNSCC treated with 5 μM YOK1104 and irradiation at 6 Gy. Scale bar: 10 μm.