spleen stomach colon - Genes & Developmentgenesdev.cshlp.org/content/suppl/2011/04/12/25.8.795.DC1/... · 2011. 4. 12. · brain heart lung spleen stomach colon kidney SM Atg5flox/flox;

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, 19M Atg5flox/+; CAG-Cre, 19M

Takamura_Fig. S2


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/+; CAG-Cre, 19MAtg5flox/flox; CAG-Cre, 19M


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


GdH

O-8

flox/flox; CAG-Cre, 6M

Takamura_Fig. S3


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


stomach colon kidney SMUb

A Atg5flox/flox; CAG-Cre, 19M


Takamura_Fig. S4

p62


stomach colon kidney SM


stomach colon kidney SMp62

B Atg5flox/flox; CAG-Cre, 19M



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


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





8-O

HdG

A

B



Takamura_Fig. S6


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


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


Total

Atg7

Takamura_Fig. S7


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.

Documents

spleen stomach colon - Genes & Developmentgenesdev.cshlp.org/content/suppl/2011/04/12/25.8.795.DC1/... · 2011. 4. 12. · brain heart lung spleen stomach colon kidney SM Atg5flox/flox;