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Takamura_Fig. S1 brain heart lung spleen stomach colon kidney SM Supplemental Figure 1 Histological findings of tg5 flox/flox ;CG-Cre mouse tissues. H&E staining of the brain, heart, lung, spleen, stomach, colon, kidney, and smooth muscle (SM) at 19 months (n=4). Scale bar=50 µm.

tg5 flox/flox ; CG-Cre, 12M tg5 flox/flox ; CG-Cre, 12M Takamura_Fig. S2 tg5 flox/flox ; CG-Cre, 6M tg5 flox/flox ; CG-Cre, 9M C Oil-red O Supplemental Figure 2 Histological findings of the liver in tg5 flox/flox ;CG-Cre mice. () Hematoxylin and eosin (H&E) staining (left, a higher magnification image shown in Figure 2) and silver impregnation staining (right) of tg5 flox/flox ;CG-Cre mouse liver at 12 months. Scale bar=500 µm. () High-power field images of H&E staining of the livers from tg5 flox/flox ;CG-Cre mice at 6 months (n=3), 9 months (n=5), and 19 months (n=4), and an tg5 flox/+ ;CG-Cre mouse at 19 months (n=4) (the last two are higher magnification images shown in Figure 2). Scale bar=20 µm. (C) Oil-red O staining of tg5 flox/flox ;CG-Cre and tg5 flox/+ ;CG-Cre mouse livers at 19 months (n=2). Scale bar=20 µm.

tg14 Flox allele Takamura_Fig. S3 Recombinant allele 12M 7d 1M 6M 9M 12M 19M 12M 7d 1M 6M 9M 12M19M CG-Cre CG-Cre Ki67 C flox/flox; CG-Cre, 6M 8-OHdG DN quantity/ Supplemental Figure 3 Growth activity and oxidative stress responses in tg5 flox/flox ;CG-Cre and control mouse liver. () Real-time PCR assay of the tg5 flox and tg5 recombinant alleles. Genomic DN was extracted from paraffin-embedded liver sections of tg5 flox/flox ;CG-Cre and tg5 flox/flox mice at different ages, as indicated. The DN quantity of the tg5 flox and recombinant alleles (relative to that of tg14 genomic DN) in tg5 flox/flox MEFs are set as 1 and 0, respectively. The quantities in tg5 / MEFs are set as 0 and 1, respectively. 7 days, n=2; 1 month, n=3; 6 months, n=2; 9 months, n=4; 12 months, n=1; 19 months, n=3. () Immunohistochemical analysis of tg5 flox/+ ;CG-Cre mouse liver at 19 months (0.4 ± 0.1%) for Ki-67 expression. n=3. Scale bar=50 µm. (C) 8-OHdG staining in the liver of tg5 flox/flox ;CG-Cre mice at 6 months (44.3 ± 4.0%)(n=2), tg5 flox/+ ;CG-Cre mice at 19 months (2.1 ± 0.5%) (n=3). Scale bar=40 µm.

Takamura_Fig. S4 Ub Ub Supplemental Figure 4 ccumuration of ubiquitin- and -positive aggregates in organs. (,) Ubiquitin () and () staining in organs from tg5 flox/flox ;CG-Cre mice (n=2) and tg5 flox/+ ;CG-Cre (n=2) mice at 19 months. Low-power (scale bar=40 µm) and high-power field images (insets, scale bar=5 µm) are shown.

Takamura_Fig. S5 GST Supplemental Figure 5 Induction of the oxidative stress in tg5 flox/flox ;CG-Cre mouse liver. GST staining in the liver of tg5 flox/flox ;CG-Cre and tg5 flox/+ ;CG-Cree mice at 19 months. n=3. Scale bar=100 µm (10 µm in insets).

Takamura_Fig. S6 8-OHdG Histone H2 Supplemental Figure 6 Induction of the oxidative stress and DN damage responses are not observed in organs other than the liver of tg5 flox/flox ;CG-Cre mice. (,) 8-OHdG () and phospho-histoneh2.x () staining in organs of tg5 flox/flox ;CG-Cre mice at 19 months. n=2. Scale bars=40 µm (), 500 µm (), and 20 µm (insets).

Takamura_Fig. S7 tg7 lb-cre_non-tumor ; tg7 lb-cre_non-tumor ; Keap1 Keap1 Nqo1 Detergent soluble Detergent insoluble Nqo1 Detergent soluble Detergent insoluble tg7 tg7 actin actin Total Total tg7 lb-cre_non-tumor ; Keap1 Nqo1 Detergent soluble Detergent insoluble tg7 actin Total Supplemental Figure 7 -dependent sequestration of Keap1 into inclusions and Nqo1 induction. Immunoblot analysis of liver samples obtained from wild type (indicated as control),, tg7 flox/flox ;lb-cre (non-tumor area and tumor area), and tg7 flox/flox ;lb-cre; mice. Liver homogenates were separated into detergent-soluble and detergent-insoluble fractions with 0.5% Triton X-100. Each fraction was subjected to SDS PGE and analysed by immunoblotting with the indicated antibodies. Nqo1; ND(P)H dehydrogenase quinone 1. The results of three independent experiments are shown.

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