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. 2021 Jan 4;12:16. doi: 10.1038/s41467-020-20185-1

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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. 8 — in vivo a Immunoblot analysis with homogenates from livers of three 5-week-old HyD-LIR^flox/flox and HyD-LIR^flox/flox; Alb-Cre mice. Data shown are representative of three separate experiments. Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to whole proteins estimated by Ponceau-S staining. Data are means ± s.e. *p < 0.05, **p < 0.01, and ***p < 0.001 as determined by two-sided Welch’s t-test. Source data are provided as a Source Data file. b Immunohistofluorescence microscopy. Paraffin sections of mouse livers described in a were double-immunostained with p62 and ubiquitin, S351-phosphorylated p62 or Keap1 antibodies. Bars: 20 µm. c Proteomics analysis with liver homogenates of mice described in a. Nrf2-target genes are indicated in yellow color. d Gene expression of Nrf2-targets. Total RNAs were prepared from mouse livers of 5-week-old HyD-LIR^flox/flox (n = 3) and HyD-LIR^flox/flox; Alb-Cre mice (n = 3). Values were normalized against the amount of mRNA in HyD-LIR^flox/flox livers. RT-qPCR analyses were performed as technical replicate on each biological sample. Data are means ± s.e. *p < 0.05, **p < 0.01, and ***p < 0.001 as determined by two-sided Welch’s t-test. Source data are provided as a Source Data file. e Liver weight (% per body weight) of 5-week-old HyD-LIR^flox/flox (n = 4), HyD-LIR^flox/flox; Alb-Cre (n = 4), Atg7 ^flox/flox (n = 4), and Atg7^flox/flox; Alb-Cre (n = 4) mice. Data are means ± s.e. ***p < 0.001 as determined by two-sided Welch’s t-test. Source data are provided as a Source Data file. f Hematoxylin and eosin staining of liver paraffin sections described in e. Bars: 50 µm. g Liver function tests of the mice used in 5-week-old HyD-LIR^flox/flox (n = 4), HyD-LIR^flox/flox; Alb-Cre (n = 4), Atg7 ^flox/flox (n = 4), and Atg7^flox/flox; Alb-Cre (n = 4) mice. The serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured. IU/L, international units/liter. Data are means ± s.e. *p < 0.05 as determined by two-sided Welch’s t-test. Source data are provided as a Source Data file. g Schematic diagram of liver pathology.