1
Endotoxin Accumulation Prevents Carcinogen-Induced Apoptosis
and Promotes Liver Tumorigenesis in Rodents
Supporting Data
Supporting Fig. 1. Antibiotics treatment inhibits liver fibrogenesis induced by
DEN in rats, related to Figure 1.
(A) Histological manifestation of DEN-induced hepatocarcinogenesis in rats at the
indicated times.
2
(B) Antibiotic alone caused no phenotypic manifestation in the liver of rat.
(C) Picrosirius red staining and immunostaining for α-SMA in livers from
DEN-treated rats with or without antibiotics treatment at the indicated time. Scale
bar=100μm.
(D) TUNEL assay of tumors from the DEN and antibiotics+DEN group.
3
4
Supporting Fig. 2. Macrophage infiltration is lower in TLR4-/- tumors than in wt
tumors related to Figure 2.
(A) ALT levels in tumor bearing mice of different genotypes. *, P < 0.05.
(B) Immunostaining of F4/80 positive cells in wt and TLR4-/- tumors. Scale
bar=50μm.
(C) Picrosirius red staining and immunostaining for α-SMA in tumors and
non-tumors from wt mice. Scale bar=50μm.
5
Supporting Fig. 3. Loss of TLR4 exacerbates DEN-induced liver injury, related
to Figure 3.
Representative photomicrographs (H&E stain) of livers from wt and TLR4-/- mice at 0,
24, and 48 h after DEN administration. Scale bars = 100 μm.
6
Supporting Fig. 4. TLR4 deficiency does not affect DEN-induced p53 activation
and DEN metabolism, related to Figure 3.
Mice were treated with DEN, and at the indicated time, their livers were removed for
RNA extraction. The expression levels of p53-regulated genes and DEN metabolic
enzymes mRNA were determined by qPCR
7
Supporting Fig. 5. TLR4 expression on Kupffer cells from bone marrow
transplanted mice by flow cytometry, related to Figure 4.
8
Supporting Fig. 6. BHA treatment protects TLR4-/- mice from DEN induced liver
injury, related to Figure 5.
(A) ChIP done with chromatin from DEN-treated livers using p65-specific antibody
or preimmune mouse IgG as a negative control for precipitation. Analysis was
done using specific primers for the promoter region of MnSOD, A20 and Bcl-xl.
(B) Histological analysis of the livers from mice pretreated with or without BHA
after DEN administration. Scale bar=100μm(upper panel).TUNEL staining of the
livers. Scale bar=50μm (lower panel).
9
Supporting Fig. 7. LPS/TLR4 protects against DEN-induced apoptosis, related to
Figure 6.
(A) TUNEL assay of livers DEN treated chimeric mice 48h after DEN injection.*,
P<0.05 vs other groups.
(B) Liver mRNA prepared at the indicated times after DEN injection were analyzed
by qPCR for TLR4 levels. *, P < 0.05 vs untreated mice.
10
Supporting Fig. 8. Antibiotics treatment does not affect DEN metabolism,related
to Figure 7.
(A) TUNEL assay of livers from DEN treated mice with or without antibiotics
treatment 48h after DEN injection. *, P<0.05.
(B) DEN induced p53, Mdm2 and CYP2E1 production in the livers from antibiotics
treatment mice and controls.
11
Supporting Table 1. Real-Time PCR Primer Sequences.
Gene Name
Sense
Antisense
mnsod
acaaacctgagccctaagggt
gaaccttggactcccacagac
cuznsod
aaccagtt gtgttgtcag gac
gaatg ctctc ctgagagtgag
p53
gcgtaaacgcttcgagatgtt
tttttatggcgggaagtagactg
gadd45
ccgaaaggatggacacggtg
ttatcggggtctacgttgagc
mdm2
tgtctgtgtctaccgagggtg
tccaacggactttaacaacttca
p21
cgagaacggtggaactttgac
cagggctcaggtagaccttg
bax
ccggcgaattggagatgaact
ccagcccatgatggttctgat
tnf (mice)
aagcctgtagcccacgtcgta
ggcaccactagttggttgtctttg
il-6 (mice)
tagtccttcctaccccaatttcc
ttggtccttagccactccttc
tnf (rat)
caccacgctcttctgtctactgaac
ccggactccgtgatgtctaagtact
il-6 (rat)
taccccaacttccaatgctc
ttgccgagtagacctcatagtg
18s (rat)
cggctacccacatccaaggaa
gctggaattaccgcggct
a20
tgggtgcccttttactttgaat
gctctgctgtagtccttttgaaa
bcl-xl
tgagcaggtagtgaatgaac
taggtggtcattcagatagg
-actin
agtgtgacgttgacatccgt
gcagctcagtaacagtccgc
cyp2a5
atgctgacctcaggactcctc
ggtagatggtgaatacaggacca
cyp2e1
catcaccgttgccttgcttg
gccaacttggttaaagacttggg
tctgatcatggcactgttcttc
gataaatccagccactgaagtt
gcaacttggacctg
atctgtgagcgtgtat
ggtattgaacaaggaccaagg
gaggtggagaaaggccgggt
tlr4 (for
qPCR)
tlr4 (for
genotyping)
ptpn6
12
Supporting Experimental Procedures
Tumor Induction and Analyses in Rats
Hepatocarcinogenesis in rats was chemically induced by weekly intraperitoneal (i.p.)
administration of DEN (70 mg/kg body weight; Sigma-Aldrich Inc., St. Louis, MO) over
10 weeks. At the indicated times, rats were sacrificed after ether anesthesia, and the
livers
were
immediately
removed,
weighed,
and
placed
in
ice-cold
phosphate-buffered saline (PBS). The Externally visible tumors (≥ 1 mm) were
counted and measured by stereomicroscopy. Tumor size was measured using a vernier
caliper.
Parts of the livers were fixed in 4% paraformaldehyde overnight and
paraffin embedded for histological evaluation. For antibiotic treatment, polymyxin B
(1 g/L; Fluka, Buchs, Switzerland) and neomycin (3 g/L; Sigma-Aldrich, Saint Louis, MI)
were added to the drinking water to prevent the growth of bacteria, the main source of
endotoxin in the gastrointestinal tract (1) . The antibiotic regimen started four days
prior to DEN injection until three weeks after, followed by a week of regular water.
This cycle (three weeks of antibiotics and one week of regular water) was repeated
until the rats were sacrificed.
Measurement of Hepatic Fibrosis.
4μm paraffin embedded tissue sections were stained with picrosirius red or
immunostaining for α-SMA (sigma).
Induction of Liver Tumor and Injury in Mice
For tumor induction, 12-15 day old mice and littermates were injected i.p. with 25
mg/kg DEN in sterile PBS. After ten months on normal chow, all mice were
13
sacrificed, and their livers removed and separated into individual lobes. Externally
visible tumors (≥ 0.5 mm) were counted and measured by stereomicroscopy. Tumor
size was measured using a vernier caliper. For short-term studies of DEN-induced
inflammation and liver injury, six or eight week old mice, as indicated, were injected
i.p. with DEN (100 mg/kg body weight) and sacrificed at the indicated times. For
antioxidant treatment, mice were fed on a diet containing 0.7% BHA (Sigma) for two
days and then injected with DEN.
Biochemical Analyses
Protein lysates and RNA were prepared from primary hepatocytes or liver samples for western
blotting or quantitative PCR analyses, respectively. Antibodies specific for phosphorylated Jnk,
Erk, as well as total Erk, Jnk were all purchased from Cell Signaling Technology (Danvers, MA).
Real-time quantitative PCR for analysis of mRNA expression was performed as described (2).
Primer sequences are listed in Table S1. The levels of GSH and MDA were measured using assay
kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), according to the
manufacturer’s instructions. Transaminase levels were determined by standard procedures.
Densitometric quantitation was performed using the Quantity One software.
Endotoxin Assay
Blood was taken from the abdominal aorta in pyrogen-free heparinized syringes
during laparotomy at indicated time after DEN administration. Plasma endotoxin was
measured using the Limulus amebocyte lysate pyrogen test (Xiamen Houshiji, Ltd.,
Xiamen, China). Levels of endotoxin in plasma from normal rats or mice were all
below the limit of detection.
14
Chromatin Immunoprecipitation (ChIP) Assay
The ChIP assay for analysis of NF-κB transcriptional activity was performed as described (3).
Mouse monoclonal antibody specific for p65 (Santa Cruz Biotechnology, Santa Cruz, CA)
was used. Specific primer pairs for the MnSOD, A20 and Bcl-xl promoter regions were
used for investigating the binding of NF-κB to DNA. The primer pairs are as follows:
MnSOD: sense—ccataattctgaccagcagcag, antisense--cctgtgccaaattggtagagg; A20:
sense--
gatgtgacgtggaaggcagc,
antisense--
cggctccgaagactacagactg;
Bcl-xl:
sense--gaatggaggacctggccgtc, antisense-- cagcctcggatctgtgatctg.
Flow Cytometry Analysis
Expression of TLR4 on Kupffer cells was determined by flow cytometry The hepatic
mononuclear cells were isolated and then double labeled with anti-TLR4-PE mAb
(BioLegend, San Diego, CA) and anti-CD11b-APC mAb (eBioscience, San Diego,
CA). Stained cells were assessed on a Moflo-XDP system (Beckman Coulter,
Fullerton, CA) equipped with Summit 5.1 Software.
Gut Sterilization
Mice were treated with ampicillin (1 g/L; Sigma-Aldrich), neomycin sulfate (1 g/L),
metronidazole (1 g/L; Sigma-Aldrich), and vancomycin (500 mg/L; Seishin
laboratories, Japan) in drinking water for four weeks. This treatment was followed by
DEN injection and continuation of antibiotic treatment until mice were killed (4).
15
Supporting References
1. Adachi Y, Moore LE, Bradford BU, Gao W, Thurman RG. Antibiotics prevent liver injury in
rats following long-term exposure to ethanol. Gastroenterology 1995;108(1):218-224.
2. Kong XN, Yan HX, Chen L, Dong LW, Yang W, Liu Q, et al. LPS-induced down-regulation
of signal regulatory protein {alpha} contributes to innate immune activation in macrophages.
J Exp Med 2007 Oct 29;204(11):2719-2731.
3. Yan HX, Yang W, Zhang R, Chen L, Tang L, Zhai B, et al. Protein-tyrosine phosphatase
PCP-2 inhibits beta-catenin signaling and increases E-cadherin-dependent cell adhesion. J
Biol Chem 2006 Jun 2;281(22):15423-15433.
4. Rakoff-Nahoum S, Paglino J, Eslami-Varzaneh F, Edberg S, Medzhitov R. Recognition of
commensal microflora by toll-like receptors is required for intestinal homeostasis. Cell
2004 Jul 23;118(2):229-241.