Autophagy-Dependent Generation of Axin2+ Cancer Stem-like Cells Promotes

Hepatocarcinogenesis in Liver Cirrhosis

Jun Li1*, ShaoBo Hu2*, LiYu Wang2, Xi Zhang3, Xing Zhou2, Bin Yang4, JiaHuan

Li5, Jun Xiong2, Nan Liu1, Yuan Li1, YongZhong Wu6, QiChang Zheng2

Supplementary Materials and Methods

Materials and Methods

Human Tissue Samples

A total of 8 non-liver cirrhosis surgical samples and blood samples were collected

from patients with hepatic hemangioma; 18 alcohol-related, 30 chronic hepatitis B-

related and 18 chronic hepatitis C-related liver cirrhosis biopsied samples and blood

samples were collected at Union Hospital, Tongji Medical College, Huazhong

University of Science and Technology from 09/2007. Those patients were followed-

up to 12/2015. Three surgical hepatocarcinoma samples from patients with alcohol-

related liver cirrhosis, eight hepatocarcinoma samples from those with hepatitis B-

related liver cirrhosis and four hepatocarcinoma samples from those with hepatitis C-

related liver cirrhosis were collected by surgery. These tissue samples were

immediately used for subsequent experiments or were fixed in paraformaldehyde and

paraffin-embedded after biopsy or surgical removal. Histopathological diagnosis of

hepatocarcinoma was performed according to World Health Organization criteria.

Fibrolamellarhepatocarcinoma, cholangiocarcinoma and combined

hepatocholangiocarcinoma were excluded. This study was approved by the Ethics

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Committee of Union Hospital, Tongji Medical College, Huazhong University of

Science and Technology. Written informed consent was obtained from each patient.

Generation of Transgenic Rats

To trace the origin of Axin2+CD90+ cells, an Axin2Cre rat whose Cre expression was

controlled by Axin2 promoter was crossed with a ROSA26R-EGFP reporter rat to

generate an Axin2Cre;Rosa26EGFP rat (Biocytogen, Beijing, China). In

Axin2Cre;Rosa26EGFP rat, Cre cut the termination sequence between loxP sites in

ROSA26R-EGFP and was labled by EGFP. Axin2+ cells were labled by EGFP

through Cre expression. Briefly, a PacI-BamHI fragment of pGC-Cre with a nuclear

localization signal and Cre cDNA were inserted into pMD19-T containing a mouse

Axin2 promoter fragment. After DNA sequence confirmation, this pAxin2-Cre vector

was electroporated into Sprague-Dawley rat embryonic stem cells. Following PCR

confirmation, the transgenic pAxin2-Cre embryonic stem cell clone was used to

generate germline chimeras. On the other hand, a polyadenylation signal and

neomycin resistance cassette flanked by 3’ loxP site was inserted into EcoRI and KpnI

restriction sites of pCAG-EGFP, and then the vector was inserted into fragment with

Rosa26 locus flanked by 5’ loxP site. After DNA sequence confirmation, this

expression vector was electroporated into Sprague-Dawley rat embryonic stem cells.

Following selection with G418 and PCR confirmation, the transgenic embryonic stem

cell clone carrying the ROSA26R EGFP reporter was used to generate germline

chimeras. The above heterozygous pAxin2-Cre SD rats were crossed with the

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ROSA26R rat strain containing the EGFP reporter. EGFP expression via the

transgenic promoter was visible in livers under a fluorescence microscope.

Generation of Lentiviral shRNA Vectors

The DNA fragment containing the mouse albumin promoter (–190 to +5, GenBank

accession no. M14768.1) was amplified from a male BALB/c mouse liver genomic

library by PCR and cloned into plasmid pMD19-T. After sequencing, the DNA

fragment with the mouse albumin promoter replaced the ubiquitin promoter of the

lentiviral vector pGC-lentivirus (Ltv) to generate pAlbumin-Ltv.

The Atg3, Atg7 and HGF-specific shRNAs were designed and synthesized by

GenePharma (Shanghai, China), according to the cDNA sequences in GenBank

(NM_022488, NM_006395 and NM_017017.2). The shRNA sequences were as follows:

sense: 5’-GGGAAAGGCACUGGAAGUG-3’, loop –TTCAAGA-, antisense: 5’-

CACUUCCAGUGCCUUUCCC-3’ for Atg3; sense: 5’-

ACUAAAAGGGGCAAACUGC-3’, loop –AAGTTCTCT-, antisense: 5’-

GCAGUUUGCCCCUUUUAGUAG-3’ for Atg7; sense: 5’-

AUCCUUUCAAUAGUAUGUCAAGU-3’, loop –AAGTTCTCT-, antisense: 5’-

ACUUGACAUACUAUUGAAAGGAUGC-3’ for HGF; and sense: 5’-

UCAGACAUGCAACGUCAGCU-3’, loop –TTCAAGA-, antisense: 5’-

AGCUGACGUUGCAUGUCUGAUA-3’ for the control shRNA. The shRNAs were

linked to the HpaI and XhoI restriction sites at the 5’ and 3’-ends, respectively. After

digestion, the shRNA-Atg3, shRNA-Atg7, shRNA-HGF and shRNA-control

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constructs were cloned into pAlbumin-Ltv to generate pAlbumin-Ltv-shRNA-Atg3,

pAlbumin-Ltv-shRNA-Atg7, pAlbumin-Ltv-shRNA-HGF and pAlbumin-Ltv-

shRNA-vector plasmids, respectively. Subsequently, individual recombinant lentiviral

plasmids were cotransfected with the packaging plasmids pHelper 1.0 and pHelper

2.0 into 293T cells using Lipofectamine 2000. The lentivirus virions were purified by

centrifugation and affinity chromatography. The functional viral titers of these

lentiviruses were determined by quantitative real-time PCR.

Animal Models

To establish a model of the progression of liver cirrhosis to hepatocarcinoma, 8-week-

old male Axin2Cre;Rosa26EGFP SD rats were injected intraperitoneally with 50

mg/kg diethylinitrosamine (N0258-1G, Sigma-Aldrich, Shanghai, China) twice per

week for 8 weeks. The rats were fed a regular diet for an additional 4 weeks. The rats

were biopsied to evaluate the presence of liver cirrhosis and autophagy status. Rats

with non-autophagic liver cirrhosis were intraperitoneally injected with rapamycin

(2mg/kg, S1039, Selleck, Shanghai, China) twice per week for 4 weeks. Rats with

aberrant autophagic liver cirrhosis were then number randomized and injected

intravenously with 1×109 TU/mL shRNA-vector, shRNA-Atg3, shRNA-Atg7, and

shRNA-HGF lentiviruses in transduction enhancement reagent (EnvirusTM, Engreen

Biosystem, Beijing, China) twice per week for 4 weeks. In addition, the rats also

received chloroquine (50mg/kg, an inhibitor of the late stage of autophagy, Sigma-

Aldrich) weekly, with SP600125 (a JNK inhibitor, 15 mg/kg, S1460, Selleck) or

stattic (a STAT3 inhibitor, 2 mg/kg, S7024, Selleck) administered twice per week by

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intraperitoneal injection for 4 weeks. Blood samples were then collected from each

group. A group of normal rats or rats with vehicle injection served as the control

where appropriate. The rats were sacrificed after an additional 4-week observation

after the final treatment. The entire experimental period was 20 weeks.

Liver samples from each group were collected at 4, 8 and 12 weeks after the last

diethylinitrosamine injection. The samples were immediately used for subsequent

experiments or were fixed in paraformaldehyde and paraffin-embedded for sectioning.

The animal experiments were conducted according to the guidelines for the Care and

Use of Laboratory Animals of the NIH, and the protocols were approved by the

Animal Care and Use Committee of Tongji Medical College.

Cytokine Detection

HGF, IL-2R, IL-6, IL-16, IL-18 and CXCL-12 levels in serum from individual

patients and rats were analyzed by ELISA using specific kits according to the

manufacturers’ instructions (Boster, Wuhan, China). Briefly, individual blood

samples were centrifuged at 14000 rpm for 10 min. The sera were then collected to

measure each cytokine in triplicate.

Flow Cytometry

At 4 weeks after the last diethylinitrosamine injection and 4 weeks after the last

treatment (8 weeks after the last diethylinitrosamine injection), rat liver samples were

collected. A part of each fresh human and rat liver tissue was cut into 1 mm3 pieces

and digested with collagenase IV (250 units/mL in phosphate-buffered saline [PBS]

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with 10% fetal bovine serum [FBS]) for 30 min at 37℃. The suspension was filtered

through 300 nylon mesh and centrifuged at 400 g for 5 min. Rat cell samples were

resuspended and stained with phycoerythrin (PE)-conjugated anti-CD90 (0.3μg/107

cells, 205903, Biolegend, CA, USA), anti-CD133 (0.5μg/107 cells, 130-102-834,

Miltenyi Biotec, Germany) and anti-Epcam (0.5μg/107 cells, 130-102-967, Miltenyi

Biotec) for 30 min. Human cell samples were stained with PE-conjugated anti-CD90

(0.3μg/107 cells, 328110, Biolegend), anti-CD133 (0.5μg/107 cells, 130-080-801,

Miltenyi Biotec) and anti-Epcam (0.5μg/107 cells, 130-110-999, Miltenyi Biotec)

together with fluorescein isothiocyanate (FITC)-conjugated anti-Axin2 (1.0μg/107

cells, ABIN2207368, antibodies-online, China). Control cells were stained with

isotype-matched IgG. The percentages of human Axin2+CD90+, Axin2+CD133+ and

Axin2+Epcam+ cells or rat EGFP+CD90+, EGFP+CD133+ and EGFP+Epcam+ cells

were determined by flow cytometry using BD Accuri™ C6 (BD Biosciences, San

Jose, CA, USA). The sorting of Axin2+CD90+, Axin2+CD90- and Axin2-CD90-

cells and EGFP+CD90+, EGFP+CD90- and EGFP-CD90- cells, was performed using

a commercial sorter (FACSAria, BD Biosciences), which was also used to evaluate

sphere and tumor xenograft formation capabilities. Axin2+ and Axin2- cells from

each type of human cirrhotic liver, along with EGFP+ and EGFP- cells from rat

cirrhotic livers with or without treatment, were sorted and further analyzed in vitro.

Tumor Formation Assay

Six-week-old male nude mice were from the Center of Experimental Animals (Tongji

Medical College, Huazhong University of Science and Technology, China) and were

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housed in a specific pathogen-free facility with free access to autocleaved water and

food. Individual nude mice were injected subcutaneously with 5×105 human

Axin2+CD90+, Axin2+CD90- and Axin2-CD90- cells or rat EGFP+CD90+,

EGFP+CD90- and EGFP-CD90- cells, respectively. The formation and growth of

implanted tumors were monitored for 8 weeks. The mice were then sacrificed, and the

tumor xenografts were dissected and compared in volumes. The experimental

protocols were approved by the Animal Care and Use Committee of Tongji Medical

College.

Sphere Formation Assay

The sorted human Axin2+CD90+, Axin2+CD90- and Axin2-CD90- cells and the rat

EGFP+CD90+, EGFP+CD90- and EGFP-CD90- cells, were cultured a density of

1000 cells/well in DMEM/F12 medium (HyClone, Utah, USA) supplemented with

2% B27 (Invitrogen, MA, USA), 100 U/mL of penicillin and 100 µg/mL of

streptomycin, 20 ng/mL epidermal growth factor and 20 ng/ml basic fibroblast growth

factor (bFGF, PeproTech, Rocky Hill, NJ, USA) using ultra-low attachment 6-well

plates (Corning, NY, USA) for 2 weeks. The cells were exposed to fresh medium

every 3 days. The formed spheres with diameters of>75 μm were counted.

Western Blotting

LC3-I / LC3-II conversion, P62, DNMT1 and HGF expression were detected by

Western blotting in above sorted cells and in different human and rat liver samples

with or without treatment. Western blotting was also performed to detect the

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expression of Sox2 and Oct4, β-catenin and CD90 and the phosphorylation of Met,

JNK and STAT3 in the sorted cells described above. The interference efficiencies of

Atg3 and Atg7 were confirmed by Western blot analysis in rat cirrhotic livers

transfected with shRNA-lentiviruses. Briefly, individual tissue sample or cells were

homogenized and lysed in radioimmunoprecipitation assay (RIPA) buffer

supplemented with protease inhibitor cocktail (Roche, Branford, CT, USA). The

tissue lysates (30 μg/lane) were separated by sodium dodecyl sulfate–polyacrylamide

gel electrophoresis on 12% gels and transferred to nitrocellulose membranes (Pierce,

Thermo Fisher Scientific, Waltham, MA, USA). After blocking with 5% fat-free dry

milk in Tris-buffered saline-Tween (TBST) buffer, the membranes were incubated

with primary antibodies against LC3 (1:1000; 4108, Cell Signaling Technology,

Beverly, MA, USA), p62 (1:1000; 5114, Cell Signaling Technology), p-Met

(Tyr1234/1235) (1:1000; 3077, Cell Signaling Technology), Met (1:1000; 3127, Cell

Signaling Technology), p-STAT3 (Tyr705) (1:1000; 9145, Cell Signaling

Technology), STAT3 (1:1000; 9319, Cell Signaling Technology), p-JNK (Tyr185)

(1:1000; 9255, Cell Signaling Technology), JNK (1:1000; 9252, Cell Signaling

Technology), DNMT1 (1:1000; ab188453, Abcam, MA, USA), HGF (1:1000;

ab83760, Abcam), β-catenin (1:2000; ab32572, Abcam), Atg3 (1:2000; ab108251,

Abcam), Atg7 (1:2000; ab133528, Abcam), Sox2 (1:1000; ab97959, Abcam), Oct4

(1:1000; ab18976, Abcam), CD90 (1:1000; ab92574, Abcam) and β-actin (1:2000;

ab8226, Abcam). After washing, the bound antibodies were detected with horseradish

peroxidase (HRP)-conjugated secondary antibodies (Abcam) and were visualized

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using SuperSignal™ chemiluminescence reagent (Pierce). Protein expression levels

were normalized against β-actin or total signaling molecules. The relative levels of

target protein expression were analyzed by densitometric scanning using ImageJ

software.

Immunocytofluorescence

Immunocytofluorescence was performed using the sorted Axin2- and Axin2+ cells from

human cirrhotic livers and sorted EGFP- and EGFP+ cells from rat cirrhotic livers

described above. Briefly, the cells were fixed in 4% paraformaldehyde and

permeabilized with Triton-X-100. The cells were then treated with 3% hydrogen

peroxide to inactivate endogenous peroxidase. Non-specific binding was blocked in PBS

containing 10% species-appropriate normal serum for 1 hour at room temperature.

Primary Axin2 antibody (1:200; ab32197, Abcam) or CD90 antibody (1:500; ab92574,

Abcam) was incubated overnight at 4°C in a humidified chamber. The cells were

incubated with the appropriate secondary antibody and visualized by applying peroxidase

substrate in conjunction with FITC or Cy3 (Boster, Wuhan, China).

Immunohistofluorescence

The expression levels of HGF, Axin2 and CD90 in different human and rat liver

tissue samples were determined by immunofluorescence. Briefly, the paraffin sections

(4 µm) were deparaffinized and rehydrated through graded solutions of ethanol/water.

The sections were subjected to antigen retrieval using an antigen unmasking solution

(Vector) and were then blocked in PBS containing 10% species-appropriate normal

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serum for 1 hour at room temperature. The sections were incubated with primary

antibodies against HGF (1:100; ab83760, Abcam), Axin2 (1:100; ab32197, Abcam)

and/or CD90 (1:200; ab92574, Abcam) overnight at 4°C in a humidified chamber.

The appropriate isotype-matched IgG (Vector) served as negative control. After

washing, the bound antibodies were detected with FITC- or Cy3-conjugated

secondary antibodies (1:200; Cell Signaling Technology), and images were acquired

using a fluorescence microscope. For quantitative comparison of HGF expression and

ratio of Axin2/EGFP+CD90+ cells, the ratios of positively or double positively

stained areas to total traced areas were determined by an independent pathologist

blinded to the experimental design and expressed as a percentage on high-power

(×200) images using color segmentation in Image-Pro Plus (Media Cybernetics).

Immunohistochemistry

The expression levels of α-fetoprotein (AFP) and cytokeratin 19 (CK19) in individual

tumor xenograft samples were detected by immunohistochemistry. Briefly, paraffin-

embedded sections (4 µm) were prepared as described above for

immunofluorescence, and incubated with primary antibodies against α-AFP (1:100;

ab46799, Abcam) and CK19 (1:100; ab52625, Abcam, USA) overnight at 4°C in a

humidified chamber using isotype-matched IgGs (Vector) as negative controls. The

bound antibodies were detected using biotinylated secondary antibodies and incubated

with HRP-Streptavidin (Boster, Wuhan, China) at 37°C for 20 min. After washing,

specific expression was visualized using a yellow diaminobenzidine reagent kit

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(Boster) according to the manufacturer’s instructions, and specimens were

counterstained with hematoxylin.

Histology

At 4 weeks after the last treatment (12 weeks after the last diethylinitrosamine

injection), 4-μm-thick cross-sections of rat cirrhotic livers with different autophagy

status and different treatments were cut and transferred onto gelatin-coated glass

slides. Six cross-sections from each animal were selected at 250-μm intervals for

hematoxylin-eosin (HE) and Sirius staining. Briefly, the paraffin sections were

deparaffinized and rehydrated through graded solutions of ethanol/water. The sections

were then stained with hematoxylin followed by 0.5% alcoholic eosin-Y (Sigma-

Aldrich, Shanghai, China) or with Sirius followed by hematoxylin (Sigma-Aldrich,

Shanghai, China).

Statistical Analysis

Sample size was computed by using formulas for 2 proportions comparison available

on website: http://powerandsamplesize.com/Calculators/. All data are presented as the

means ± SEM. After demonstration of homogeneity of variance with the Bartlett test,

one-way ANOVA, followed by the Student-Newman-Keuls test where appropriate,

was used to evaluate statistical significance. Values of P<0.05 were considered

statistically significant. All experiments were performed in triplicate.

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Supplementary Figures and Figure legends

Supplemental Figure I. Densitometric analysis of the Western blots shown in

Figure 1. (A) Densitometric analysis of LC3-II and P62 expression normalized to β-

actin in human normal livers and in alcohol- (n=6), HBV- (n=10) and HCV-related

cirrhotic liver tissues (n=6). (B) Densitometric analysis of LC3-II and P62 expression

normalized to β-actin in Axin2Cre;Rosa26EGFP rat normal livers and cirrhotic liver

tissues (n=4). Data are expressed as the means ± SEM of each group from three

separate experiments. *p<0.05 vs. the normal liver tissues.

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Supplemental Figure II. Quantitation of the immunofluorescence staining shown

in Figure 1B. The percentages of double positive areas (yellow) to overall areas in

individual samples were calculated. (A) The percentages of Axin2 and CD90 double

positive hepatic cells in human normal livers and in alcohol- (n=6), HBV- (n=10) and

HCV-related cirrhotic liver tissues (n=6). (B) The percentages of EGFP and CD90

double-positive hepatic cells in Axin2Cre;Rosa26EGFP rat normal livers and cirrhotic

liver tissues (n=4). Data are expressed as the means ± SEM of each group from three

separate experiments. *p < 0.05 vs. normal liver tissues.

13

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Supplemental Figure III. Flow cytometry analysis of hepatic Axin2+CD133+ and

Axin2+Epcam+ cells in human livers. Biopsied liver samples were obtained from

subjects with healthy livers (n=8) and with alcohol- (n=6), HBV- (n=10) and HCV-

related liver cirrhosis (n=6) and stratified according to autophagy status. (A)

Representative flow cytometry analysis of hepatic Axin2+CD133+ cells and (B)

Axin2+Epcam+ cells in individual liver samples. The percentages of double positive

cells are shown in the upper-right quadrants. Data are representative flow cytometry

graphs of each group from four experiments.

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Supplemental Figure IV. Flow cytometry analysis of hepatic

EGFP(Axin2)+CD133+ and EGFP(Axin2)+Epcam+ cells in

Axin2Cre;Rosa26EGFP rat livers. The biopsied rat cirrhotic liver samples were

stratified according to autophagy status. The percentages of hepatic (A)

EGFP(Axin2)+CD133+ and (B) EGFP(Axin2)+Epcam+ cells in normal livers and

cirrhotic livers with or without autophagy were determined by flow cytometry. The

percentages of double-positive cells are shown in the upper-right quadrants. Data are

representative flow cytometry graphs of each group (n=8) from three separate

experiments.

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Supplemental Figure V. Transfection with lentiviruses expressing Atg3/Atg7-

shRNAs effectively silences Atg3 and Atg7 expression in transgenic rat livers.

Axin2Cre;Rosa26EGFP rats with aberrant autophagic liver cirrhosis at 4 weeks after

induction were infected with shRNA-vector lentiviruses or lentiviruses carrying

Atg3/Atg7-shRNAs. Four weeks after transfection, Atg3 and Atg7 expression levels

in the cirrhotic liver tissues were determined by Western blot analysis. (A)

Representative Western blots for Atg3 and Atg7. (B) Densitometric analysis of Atg3

and Atg7 expression normalized to β-actin. Data are representative images or

expressed as the means ± SEM of each group (n=4) from three separate experiments.

*p < 0.05 vs. rats infected with shRNA-vector.

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Supplemental Figure VI. Densitometric analysis of the Western blots shown in

Figure 4. (A) Densitometric analysis of HGF expression normalized to β-actin in

human normal livers and in alcohol- (n=6), HBV- (n=10) and HCV-related cirrhotic

liver tissues (n=6). (B) Densitometric analysis of LC3-II, P62, DNMT1 and HGF

expression normalized to β-actin in Axin2Cre;Rosa26EGFP rat normal livers, non-

autophagic cirrhotic livers with rapamycin and aberrant autophagic cirrhotic livers

transfected with a variety of shRNAs or chloroquine (n=4). Data are expressed as the

means ± SEM of each group from three separate experiments. *p < 0.05 vs. normal

liver tissues.

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Supplemental Figure VII. Quantification of the HGF immunofluorescence

staining shown in Figure 4.

The percentages of positive HGF staining in comparison with the overall areas in

individual samples were calculated. (A) Quantitative analysis of HGF

immunofluorescence staining in human normal livers and in alcohol- (n=6), HBV-

(n=10) and HCV-related cirrhotic liver tissues (n=6). (B) Quantitative analysis of

HGF immunofluorescence staining in Axin2Cre;Rosa26EGFP rat normal livers, non-

autophagic cirrhotic livers with rapamycin and aberrant autophagic cirrhotic livers

transfected with a variety of shRNAs or chloroquine (n=4). Data are expressed as the

means ± SEM of each group from three separate experiments. *p < 0.05 vs. normal

liver tissues.

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Supplementary Table 1.

Progression ratio of liver cirrhosis to hepatocarcinoma in human samples

　 Liver cirrhosis with low autophagy Liver cirrhosis with aberrant autophagy

Cause alcohol related HBV related HCV related alcohol related HBV related HCV related

Group cases 14 20 13 4 10 5

Axin2+CD90+ cases 0 0 0 4 10 5

Hepatocarcinogenesis 0 0 0 3 8 4

Hepatocarcinogenesis ratio 0/14 0/20 0/13 3/4 8/10 4/5

During the follow-up period from 09/2007 to 12/1015, the numbers of Axin2+CD90+

and hepatocarcinogenesis cases as well as hepatocarciogenesis ratio were calculated

from patients who were stratified according to autophagy status in different types of

liver cirrhosis.

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Supplementary Table 2.

Progression rate of liver cirrhosis to hepatocarcinoma in Axin2Cre;Rosa26EGFP

rats

After 4 weeks of liver cirrhosis induction, the rats were stratified according to

autophagy status in liver cirrhosis. The rats were then administered with the indicated

reagents to induce or inhibit aberrant autophagy in cirrhotic livers. After 4 weeks of

treatment, the numbers of Axin2+CD90+ cases were calculated and the development

of hepatocarcinoma in individual group of rats were monitored up to 8 weeks after the

treatment.

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　 Liver cirrhosis

low

autophagy

Liver cirrhosis

aberrant autophagy

Treatment - rapamyc

in

- shRNA:vector/

vehicle

shRNA:Atg3

/7

chloroqui

ne

shRNA:HG

F

SP60012

5

statti

c

Group cases

Axin2+CD90+

cases

Hepatocarcinogene

sis

12 5

0 4

0 4

4

4

3

4

4

2

4

0

0

4

0

0

4

0

0

4

0

0

4

0

0