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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. 3 — a, b Chemical structures of free or biotinylated p62-ZZ small-molecule ligand YOK1104. c Schematic illustration of full-length and ZZ domain-deleted mutant p62 constructs. d In vitro YOK1104-Biotin pulldown assay using full-length p62-GFP or ΔZZ-p62-GFP proteins, purified from HEK293 cells after transient transfection of those expression vectors. e Immunostaining of p62 (red) and LC3 (green) in WSU12 HPV(-) HNSCC treated with vehicle control (DMSO) or 5 μM YOK1104. Scale bar: 10 μm. f Immunoblot analysis of p62, LC3, or GAPDH with or without YOK1104 treatment. g Immunostaining of LC3 (red) and LAMP1 (green) in WSU12 HPV(-) HNSCC treated with vehicle control (DMSO) or 5 μM YOK1104. Scale bar: 10 μm. h Immunostaining of Beclin1 (red) in UM19 HPV(-) HNSCC treated with vehicle control (DMSO) or 5 μM YOK1104. Scale bar: 20 μm. i Immunostaining of p62 (red) and LC3 (green) in UP090 HPV( + ) HNSCC treated with vehicle control (DMSO) or 5 μM YOK1104. Scale bar: 10 μm. j Quantification of colocalization (yellow) of p62 with LC3 in e and i (n = 20 cells) (***p < 0.001, ns non-significant). k Immunostaining of LC3 (red) and LAMP1 (green) in UP154 HPV( + ) HNSCC treated with vehicle control (DMSO) or 5 μM YOK1104. Scale bar: 10 μm. l Quantification of colocalization (yellow) of LC3 with LAMP1 in g and k (n = 20 cells) (***p < 0.001, ns: non-significant). m Immunostaining of Beclin1 (red) in UP154 HPV( + ) HNSCC treated with vehicle control (DMSO) or 5 μM YOK1104. Scale bar: 20 μm.