spleen stomach colon - genes &...
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brain heart lung
spleen stomach colon
kidney SM
Atg5flox/flox; CAG-Cre, 19M
Takamura_Fig. S1
Supplemental Figure 1
Histological findings of Atg5flox/flox;CAG-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.
A
B
C Oil-red O
Atg5flox/flox; CAG-Cre, 12M
Atg5flox/flox; CAG-Cre, 6M
Atg5flox/flox; CAG-Cre, 19M Atg5flox/+; CAG-Cre, 19M
Takamura_Fig. S2
Supplemental Figure 2
Histological findings of the liver in Atg5flox/flox;CAG-Cre mice.
(A) Hematoxylin and eosin (H&E) staining (left, a higher magnification image shown in Figure 2A) and silver impregnation staining (right) of
Atg5flox/flox;CAG-Cre mouse liver at 12 months. Scale bar=500 µm.
(B) High-power field images of H&E staining of the livers from Atg5flox/flox;CAG-Cre mice at 6 months (n=3), 9 months (n=5), and 19 months (n=4),
and an Atg5flox/+;CAG-Cre mouse at 19 months (n=4) (the last two are higher magnification images shown in Figure 2B). Scale bar=20 µm.
(C) Oil-red O staining of Atg5flox/flox;CAG-Cre and Atg5flox/+;CAG-Cre mouse livers at 19 months (n=2). Scale bar=20 µm.
Atg5flox/flox; CAG-Cre, 12M
Atg5flox/+; CAG-Cre, 19MAtg5flox/flox; CAG-Cre, 19M
Atg5flox/flox; CAG-Cre, 9M
Flox allele Recombinant allele
12M 7d 19M9M6M 12Mf/f f/f; CAG-Cre
1M 12M 7d 19M9M6M 12Mf/f f/f; CAG-Cre
1M
A76i
K
B
C
Atg5flox/+; CAG-Cre, 19M
Atg5flox/+; CAG-Cre, 19M
GdH
O-8
flox/flox; CAG-Cre, 6M
Takamura_Fig. S3
Supplemental Figure 3
Growth activity and oxidative stress responses in Atg5flox/flox;CAG-Cre and control mouse liver.
(A) Real-time PCR assay of the Atg5 flox and Atg5 recombinant alleles. Genomic DNA was extracted from paraffin-embedded
liver sections of Atg5flox/flox;CAG-Cre and Atg5flox/flox mice at different ages, as indicated. The DNA quantity of the Atg5 flox and
recombinant alleles (relative to that of Atg14 genomic DNA) in Atg5flox/flox MEFs are set as 1 and 0, respectively. The quantities in
Atg5∆/∆ 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.
(B) Immunohistochemical analysis of Atg5flox/+;CAG-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 Atg5flox/flox;CAG-Cre mice at 6 months (44.3 ± 4.0%)(n=2),Atg5flox/+;CAG-Cre mice at 19 months
(2.1 ± 0.5%) (n=3). Scale bar=40 µm.
41gtA /ytitnauq
AN
D
brain heart lung spleen
stomach colon kidney SMUb
brain heart lung spleen
stomach colon kidney SMUb
A Atg5flox/flox; CAG-Cre, 19M
Atg5flox/+; CAG-Cre, 19M
Takamura_Fig. S4
p62
brain heart lung spleen
stomach colon kidney SM
brain heart lung spleen
stomach colon kidney SMp62
B Atg5flox/flox; CAG-Cre, 19M
Atg5flox/+; CAG-Cre, 19M
Supplemental Figure 4
Accumuration of ubiquitin- and p62-positive aggregates in organs.
(A,B) Ubiquitin (A) and p62 (B) staining in organs from Atg5flox/flox;CAG-Cre mice (n=2) and Atg5flox/+;CAG-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.
GS
T
Atg5flox/flox; CAG-Cre, 19M Atg5flox/+; CAG-Cre, 19M
Takamura_Fig. S5
Supplemental Figure 5
Induction of the oxidative stress in Atg5flox/flox;CAG-Cre mouse liver.
GST staining in the liver of Atg5flox/flox;CAG-Cre and Atg5flox/+;CAG-Cree mice at 19 months. n=3. Scale bar=100 µm (10 µm in insets).
His
tone
H2A
brain heart lung spleen
stomach colon kidney SM
brain heart lung spleen
stomach colon kidney SM
8-O
HdG
A
B
Atg5flox/flox; CAG-Cre, 19M
Atg5flox/flox; CAG-Cre, 19M
Takamura_Fig. S6
Supplemental Figure 6
Induction of the oxidative stress and DNA damage responses are not observed in organs other than the liver of
Atg5flox/flox;CAG-Cre mice.
(A,B) 8-OHdG (A) and phospho-histoneH2A.X (B) staining in organs of Atg5flox/flox;CAG-Cre mice at 19 months. n=2. Scale bars=40 µm
(A), 500 µm (B), and 20 µm (insets).
.tnoC7gtA
;f/f
-blA
;erC
26p
-/-
26p
-/- 7gtA
;f/f
-blA
erC
romut-non_
7gtA
;f/f
-blA
erC
romut_
.tnoC7gtA
;f/f
-blA
;erC
26p
-/-
26p
-/- 7gtA
;f/f
-blA
erC
romut-non_
7gtA
;f/f
-blA
erC
romut_
.tnoC7gtA
;f/f
-blA
;erC
26p
-/-
26p
-/- 7gtA
;f/f
-blA
erC
romut-non_
7gtA
;f/f
-blA
erC
romut_
p62
Keap1
Nqo1
actin
Detergent soluble Detergent insoluble
Total
Atg7
.tnoC7gtA
;f/f
-blA
;erC
26p
-/-
26p
-/- 7gtA
;f/f
-blA
erC
romut-non_
7gtA
;f/f
-blA
erC
romut_
.tnoC7gtA
;f/f
-blA
;erC
26p
-/-
26p
-/- 7gtA
;f/f
-blA
erC
romut-non_
7gtA
;f/f
-blA
erC
romut_
.tnoC26p
-/- 7gtA
;f/f
-blA
erC
romut-non_
7gtA
;f/f
-blA
erC
romut_
p62
Keap1
Nqo1
actin
Detergent soluble Detergent insoluble
Total
Atg7
.tnoC7gtA
;f/f
-blA
;erC
26p
-/-
26p
-/- 7gtA
;f/f
-blA
erC
romut-non_
7gtA
;f/f
-blA
erC
romut_
.tnoC7gtA
;f/f
-blA
;erC
26p
-/-
26p
-/- 7gtA
;f/f
-blA
erC
romut-non_
7gtA
;f/f
-blA
erC
romut_
.tnoC7gtA
;f/f
-blA
;erC
26p
-/-
26p
-/- 7gtA
;f/f
-blA
erC
romut-non_
7gtA
;f/f
-blA
erC
romut_
p62
Keap1
Nqo1
actin
Detergent soluble Detergent insoluble
Total
Atg7
Takamura_Fig. S7
Supplemental Figure 7
p62-dependent sequestration of Keap1 into inclusions and Nqo1 induction.
Immunoblot analysis of liver samples obtained from wild type (indicated as control), p62-/-,
Atg7flox/flox;Alb-Cre (non-tumor area and tumor area), and Atg7flox/flox;Alb-Cre; p62-/- mice. Liver homogenates
were separated into detergent-soluble and detergent-insoluble fractions with 0.5% Triton X-100. Each
fraction was subjected to SDS–PAGE and analysed by immunoblotting with the indicated antibodies. Nqo1;
NAD(P)H dehydrogenase quinone 1. The results of three independent experiments are shown.