TLR4 Enhances TGF-! Signaling and Hepatic Fibrosis
Ekihiro Seki1,2, Samuele De Minicis1,2, Christoph H. Österreicher1,2, Johannes Kluwe1,
Yosuke Osawa1, David A. Brenner2, and Robert F. Schwabe1
SUPPLEMENTARY METHODS
Mice and Fibrosis induction
CCl4 (Sigma-Aldrich, diluted 1:3 in corn oil) or vehicle (corn-oil) was administered by
gavage at a dose of 1 µl/g body weight every five days for a total of eight injections.
TAA-induced liver fibrosis was induced by administering TAA (Sigma-Aldrich) at a dose
of 300 mg/L in drinking water for 20 weeks. Some mice were infected with adenoviruses
expressing dnBAMBI or empty Adshuttle control virus (1x109 pfu/ml in PBS i.v./mouse)
48 hours before BDL. Some mice received liposomal clodronate (200 µl i.v.) one day
before BDL to deplete KCs. Liposomal clodronate was prepared as described 1.
Microarray
Six hours after isolation, quiescent HSCs were treated with E.coli 055:B5 LPS (Sigma) at
a concentration of 100 ng/ml or vehicle (endotoxin-free H2O) for six hours (n=3 HSC
isolations). RNA was extracted by a combination of Trizol (Invitrogen) and RNeasy
columns (Qiagen). Microarray analysis was performed using mouse genome 430 2.0
array gene chips (Affymetrix) according to the manufacturer’s instructions. Briefly, first
strand and second strand synthesis were performed using the one-cycle cDNA synthesis
kit (Affymetrix) followed by cleanup of double-stranded cDNA using cDNA spin columns
(Affymetrix). Biotin-labeled cRNA was generated using one cycle IVT labeling kit
followed by cRNA cleanup using cRNA spin columns (both Affymetrix). cRNA
fragmentation which was confirmed by agarose gel electrophoresis. After hybridization to
gene chip 430, scanning with a genechip Scanner 3000 7G (Affymetrix) data analysis
were performed using Genespring GX 7.3 (Agilent). Analysis was performed as paired
analysis comparing untreated and LPS-treated HSCs from the same isolation. Genes
that displayed a significant (p<0.015) and at least 2-fold up- or downregulation were
considered differentially regulated.
Reverse transcription PCR and real time quantitative PCR
RNA was isolated from mouse HSCs by the RNeasy kit, DNAse treated and was reverse
transcribed. PCR for BAMBI was performed using primers 5’-atggatcgccactccagctac-3’
and 5’-tcatatgaattccagctttccatgac-3’ at 95°C (45s), 60°C (45s) and 72°C (90s) for 33
cycles. Actin PCR was performed using 5’-GACGATATCGCTGCGCTG-3’ and 5’-
GTACGACCAGAGGCATACAGG-3’ for 27 cycles. Real time quantitative PCR was
performed for 40 cycles of 15 seconds at 95°C and 60 seconds at 60° C using an ABI
7000 sequence detection system (Applied Biosystems) and primer-probe sets from ABI.
Quantification was performed by comparing the Ct values of each sample to a standard
curve and normalization to 18s or !-actin. Values are expressed as fold induction in
comparison to untreated or sham controls.
Adenoviral construction and purification
Adenoviruses expressing I"Bsr, GFP, beta-galactosidase and NF-"B-driven luciferase
have been described previously and were amplified as previously described 2,3. Full-
length BAMBI (cloned from murine HSC cDNA library by RT-PCR), N-terminally deleted
dominant negative BAMBI 4 or BAMBI promoter-driven luciferase were constructed
using the AdEasy system (ATCC). Briefly, dominant negative BAMBI 4 was excised from
pcDNA 3.0 using KPNI and SmaI and ligated into CMV-Track and CMV-Shuttle using
KPNI and EcoRV. Full length BAMBI was ligated into CMV-Track and CMV shuttle using
HindIII and EcoRV. The human BAMBI promoter (-3382 to +82) 5,6 and luciferase were
excised from PGL3 basic by KPNI and NcoI, and NcoI and SalI, respectively, and ligated
into Shuttle plasmid using KPNI and SalI. Linearization, recombination and adenoviral
expansion were performed as previously described 3.
NF-"B, Smad and BAMBI reporter assays
For NF-"B reporter assays, HSCs (4x105 cells/well) were co-infected with AdNF"Bluc 3
and CMV-promoter driven Ad5LacZ 3 at at a multiplicity of infection (moi) of 100
particles/cell 12 hours after isolation for 12 hours followed by stimulation with various
concentrations of LPS for six hours. For TGF! reporter assays, HSCs were co-infected
with AdCAGA-Luc 7 at an moi of 100 and Ad5Shuttle,AdGFP, AddnBAMBI or AdBAMBI
12 hours after isolation using an moi of 300. 24 hours after isolation, cells were
pretreated with LPS (100 ng/ml) for 16 hours followed by treatment with recombinant
TGF!1 (R&D systems) at 300 pg/ml for six hours. For BAMBI reporter assays, HSCs
were co-infected with AdBAMBI-Luc and Ad5LacZ at an moi of 100 12 hours after
isolation. 18 hours after isolation, cells were treated with LPS (100 ng/ml) for 24 hours.
Luciferase activity was measured in a multiwell platereader (Fluostar Optima) and
normalized to !-galactosidase as described 3.
Measurement of hepatic collagen and hyaluronan content
Hydroxyproline content was measured as previously described 8. Hepatic collagen
content was analyzed by Sirius red staining of paraffin-embedded sections 8. The Sirius
red positive area was measured in at least six low power (40x) fields on each slide and
quantified using NIH imaging software. To determine the hepatic hyaluronan content,
liver was homogenized in 10mM Tris-Hcl (pH 7.4), 1mM EDTA, 150mM NaCl. Serum
and hepatic hyaluronan content were measured by ELISA (Echelon Biosciences Inc) as
suggested by the manufacturer.
Cell migration and cell adhesion assay
Cell migration assay was analyzed by the QCM Chemotaxis Cell Migration Assay (5 µm
pore size, Chemicon International). The lower chamber was filled with conditioned
medium from HSCs stimulated with LPS (100 ng/ml) or vehicle for 48 hours. KCs from
either TLR4-wild type or TLR4-mutant mice were placed into the upper chamber (105
cells/well) and incubated at 37°C for eight hours. Adhesion of KCs to HSCs was
determined cell adhesion assay as previously described 9. Briefly, HSCs were isolated
from TLR4 wild-type mice and incubated with LPS (100 ng/ml) or vehicle for 24 hours.
KCs were isolated from either TLR4-wild type or TLR4-mutant mice and labeled by
CellTracker Green CMFDA (Molecular Probes) and placed on HSCs for one hour. HSCs
were then gently washed and adherent KCs were counted in ten randomly chosen high-
power fields.
SUPPLEMENTARY REFERENCES
1. Van Rooijen, N. & Sanders, A. Liposome mediated depletion of macrophages:
mechanism of action, preparation of liposomes and applications. J Immunol
Methods 174, 83-93 (1994).
2. Iimuro, Y. et al. NFkappaB prevents apoptosis and liver dysfunction during liver
regeneration. J Clin Invest 101, 802-11 (1998).
3. Schwabe, R.F. & Sakurai, H. IKKbeta phosphorylates p65 at S468 in
transactivaton domain 2. Faseb J 19, 1758-60 (2005).
4. Onichtchouk, D. et al. Silencing of TGF-beta signalling by the pseudoreceptor
BAMBI. Nature 401, 480-5 (1999).
5. Sasaki, T., Sasahira, T., Shimura, H., Ikeda, S. & Kuniyasu, H. Effect of Nma on
growth inhibition by TGF-betaa in human gastric carcinoma cell lines. Oncol Rep
11, 1219-23 (2004).
6. Sekiya, T. et al. Identification of BMP and activin membrane-bound inhibitor
(BAMBI), an inhibitor of transforming growth factor-beta signaling, as a target of
the beta-catenin pathway in colorectal tumor cells. J Biol Chem 279, 6840-6
(2004).
7. Dooley, S. et al. Smad7 prevents activation of hepatic stellate cells and liver
fibrosis in rats. Gastroenterology 125, 178-91 (2003).
8. Bataller, R. et al. NADPH oxidase signal transduces angiotensin II in hepatic
stellate cells and is critical in hepatic fibrosis. J Clin Invest 112, 1383-94 (2003).
9. Hellerbrand, Wang, S.C., Tsukamoto, H., Brenner, D.A. & Rippe, R.A.
Expression of intracellular adhesion molecule 1 by activated hepatic stellate cells.
Hepatology 24, 670-6 (1996).
Oil CCl4
a
!"SMA
#-Actin
Vehicle TAA
b
Siri
us r
ed-p
ositi
ve a
rea
(%)
Siri
us r
ed-p
ositi
ve a
rea
(%)
Tlr4+ Tlr4mut
!"SMA
#-Actin
0 1 2 0 1 2
Vehicle TAA Vehicle
Tlr4+ Tlr4mut
TAA
0 1 2 0 1 2
Oil CCl4 Oil
Tlr4+ Tlr4mut
CCl4
0
5
10
15
20
25
*
Tlr4+
Tlr4mut
End
otox
in (
EU
/ml)
0
c d e fshamday5day21
0Tlr4+ Tlr4mut
0.1
0.2
0.3
0.4
0.5
0.6
0
0.04
0.08
0.12
VehTAA
Sirius
red
!"SMA
Sirius
red
!"SMA
End
otox
in (
EU
/ml)
Tlr4+ Tlr4mut
0
2
4
6
8
*
Tlr4+
Tlr4mut
Control Antibiotics
End
otox
in (
EU
/ml)
00.10.2
0.3
0.40.50.6 *
shamday5day21
0.1
0.2
0.3
Oil1xCCl48xCCl4
End
otox
in (
EU
/ml)
Tlr4+ Tlr4mut Tlr4+ Tlr4mut0
Supplementary figure 1. Hepatic fibrogenesis and endotoxin levels in Tlr4-mutant mice.
(a) Tlr4-mutant C3H/HeJ (n=6) and Tlr4-wild type C3H/HeOuJ (n=6) mice were treated with 8gavages of CCl4 (1 µl/g). Hepatic fibrosis was assessed by Sirius red staining, !-SMA
immunohistochemistry and !-SMA western blot. Scale bar 50 µM. (b) Tlr4-mutant C3H/HeJ
(n=6) and Tlr4-wild type C3H/HeOuJ (n=6) mice were treated with TAA (300 mg/L) in drinkingwater for 20 weeks. Hepatic fibrosis was assessed as described above. Scale bar 50 µM. *
p<0.05 for CCl4- or TAA-treated Tlr4-mutant mice versus CCl4- or TAA-treated Tlr4-wild type
mice. (c-f) Plasma endotoxin levels were measured by Limulus endotoxin assay in Tlr4-wild
type and Tlr4-mutant mice following BDL (c), CCl4 (d), TAA (e) or BDL plus antibiotics cocktail
(f). * p<0.05 control-treated mice vs. antibiotics treated mice after 21 days of BDL.
sham BDL sham BDL sham BDL Sham BDL
a b
c
0
5
10
15
20
0
2
4
6
8
0
1
2
3
4
*
0
20
40
60
Tlr2–/–
Sham BDL0
5
10
15
BDL BDL
shamsham
Siri
us r
ed-p
ositi
ve a
rea
(%)
Col
1a1
mR
NA
Act
a2 m
RN
A
Tgf
b1 m
RN
A
Tim
p1 m
RN
A
Tlr2–/–
Tlr2+/+
Tlr2–/–
Tlr2+/+
Tlr2+/+
Supplementary figure 2. TLR2 is dispensable for hepatic fibrosis. Tlr2-deficient mice and
wild-type mice underwent BDL and were sacrificed after 5 days (n=5 mice each group) and
21 days (n=8 each group) or sham operation for the same duration (n=2 each group). (a-b)Hepatic fibrosis was assessed by Sirius red staining 21 days after BDL. Scale bar 50 µM. (c)
Col1a1, Acta2, Tgfb1 and Timp1 mRNA levels were determined by qPCR 5 days after BDL. *
p< 0.05 for bile duct ligated Tlr2-deficient mice versus bile duct ligated wild type mice.
a
b
Control Clodronate
ControlClodronate
Sham BDL Sham BDL Sham BDL Sham BDL0
20
40
60
*
0
10
20
*
0
2
4
6
8
*
0
100
200
300
400
*
Control Clodronated
Sham BDL0
100
200
300
400
*
Sham BDL
*
0
5
10
15
20
Hyd
roxy
prol
ine
mg/
g liv
er
Siri
us r
ed-p
ositi
ve a
rea
(%)
Control Clodronate
BDL
Sham Sham
BDL
!"SMA
#-Actin
0 1 2
Sham BDL
Control
3
Clodronate
Sham BDL
0 1 2 3
f
e
g
BDL
Sham Sham
BDL
Ser
um A
LT (
IU/li
ter)
0
200
400
600
800
1000
1200
Sham5 days
KC
s C
lodr
onat
e vs
. con
trol
(%
)
0Control Clodronate
20
40
60
80
100
*
120
BDL BDL21 days
Col
1a1
mR
NA
Act
a2 m
RN
A
Tgf
b1 m
RN
A
Tim
p1 m
RN
A
ControlClodronate
Supplementary figure 3. KCs are required for hepatic fibrogenesis. Wild-type C57Bl/6
mice were injected with liposomal chlodronate (200 µl, i.v., n=5 for each time point) or
vehicle (n=5 for each time point). (a) Immunofluorescent staining for F4/80 (green) and
desmin (red) was performed to detect effects of clodronate on KC and HSC number,
respectively. Scale bar 10 µM. (b) Col1a1, Acta2, Tgfb1 and Timp1 mRNA levels were
determined by qPCR 5 days after BDL and are expressed as fold-induction. (c) Hepatic
injury was assessed by serum ALT levels in control and KC-depleted mice. (d-g) Hepatic
fibrosis was determined 21 days after BDL by Sirius red staining (d, f), !-SMA
immunohistochemistry (e), hepatic hydroxyproline content (f) and !-SMA western blot (g).
Scale bar 50 µM. *p<0.05 for KC-depleted mice versus control mice after BDL.
c
[3H
] T
hym
idin
e [cpm
]
0
2000
4000
6000
8000
10000
12000
14000
16000
PDGF LPS
– + +– – +
*
n.s.
LPS (100 ng/ml) – + – + – + – + – +
PDGF (ng/ml) 0 0.015 0.625 2.5 10
phospho Akt
total Akt
-
total Erk
-phospho Erk
ab
--
CA
GA
rep
orte
r ac
tivity
0
3
6
9
12
15
- - + - +LPS (100 ng/ml)
0 0.3 0 0.3 0.3 1 3 0.3 1 3
mR
NA
0
1
2
3
4
Smad2 Smad3 Smad4 Smad7 Tgfbr1 Tgfbr2 Tgfb1 Itga5 Itgb6
VehLPS
c
d
+ -- + +
TGF-# (ng/ml) latent TGF-# (ng/ml)
Supplementary figure 4. Effects of LPS on PDGF-induced signals and TGF-#
signaling pathways in HSCs. (a) Quiescent HSCs were pretreated with LPS (100 ng/ml)
or vehicle for 24 hours followed by the indicated concentration of PDGF 15 minutes.
Phosphorylation of Akt and Erk was detected by western blot. (b) Quiescent HSCs were
pretreated with LPS (100 ng/ml) or vehicle for 24 hours followed by PDGF (20 ng/ml) for 48
hours. [3H]-thymidine was added for the last 16 hours and DNA synthesis was measured in
a scintillation counter.(c) Gene expression was determined by qPCR in quiescent HSCs
treated with LPS (100 ng/ml) or vehicle for 6 hours and is expressed as fold-induction. (d)To determine potential effects of LPS on TGF-# activation, quiescent HSCs were
transduced with CAGA-luciferase followed by LPS (100 ng/ml) pretreatment for 24hfollowed by either TGF-#1 (0.3 ng/ml) or latent TGF-#1 (0.3, 1 or 3 ng/ml) for 8 hours.
CAGA-driven luciferase activity is expressed as fold induction after normalization to #-
galactosidase activity.
-
-
Bambi
#-actin
HSC KC Hepatocytes
Ad-Shuttle
Ad-dnBambi
Sham BDL Sham BDL
Col
1A1
mR
NA
Act
a2 m
RN
A
0
5
10
15
20
0
4
8
12
a b
Supplementary figure 5. dnBambi overexpression enhances hepatic fibrogenesis. (a)
Bambi mRNA expression in HSCs, KCs and hepatocytes was determined by RT-PCR. (b) Wild-
type C57Bl/6 mice were infected with empty Ad-shuttle and Ad-dnBambi (1x109 pfu i.v./mouse).
Mice underwent BDL (n=5) or sham operation (n=2) 48 hours after adenovirus inoculation.
Col1a1 and Acta2 mRNA levels were determined by qPCR 5 days after BDL and are expressed
as fold-induction. * p<0.05
Supplementary figure 6. NF-$B regulates Bambi expression and LPS-mediated
sensitization to TGF-#. (a) Quiescent HSCs (cultured for 6 hours) were infected with Ad-
Shuttle control virus or with Ad-I$Bsr (both 300 moi) and 24 hours later treated with LPS (100
ng/ml) for 6 hours. Bambi mRNA expression was determined by qPCR and is expressed as fold-
induction. (b) Quiescent HSCs from Coll-GFP (cultured for 6h after isolation) infected with emptyAd-Shuttle or with Ad-I$Bsr (both 30 moi) and 24 hours later treated with LPS (100 ng/ml) for 24
hours followed treatment by TGF-#1 (100 pg/ml) or vehicle for additional 48 hours. LPS-
mediated sensitization to TGF-# in the presence/absence of NF-$B activation was determined
by quantification of GFP expression. Scale bar 25 µM.
Co
llag
en
-GF
P+ c
ells
(%
)
TGF-# – + – +– – + +LPS
* –TGF-# +TGF-# –TGF-# +TGF-#
Ad-Shuttle Ad-I$Bsr
0
10
20
30
40
50
Control I$Bsr
*
NS
- LPS - LPS
+ LPS + LPS
- LPS - LPS
+ LPS + LPS
Bam
bi m
RN
A (
fold
indu
ctio
n)
0.0
0.5
1.0
1.5
2.0
Control I$Bsr
VehLPS
a b
– + – +– – + +
*
*
Supplementary Table 1.
Six hours after isolation, quiescent mouse HSCs were treated with LPS (100 ng/mL) for
six hours. Three different HSC isolations were used for microarray analysis. Genes that
were at least 2-fold up- or downregulated were determined as described in Materials and
Methods. Shown are 121 differentially regulated genes (corresponding to 139 probe
sets).
Probe Set ID Gene Title Gene Symbol Fold change (range)
1449984_at chemokine (C-X-C motif) ligand 2 Cxcl2 19.73 (11.68 to 40.5) 1419561_at chemokine (C-C motif) ligand 3 Ccl3 17.98 (3.269 to 47.35) 1418930_at chemokine (C-X-C motif) ligand 10 Cxcl10 15.94 (13.08 to 19.38) 1421712_at selectin, endothelial cell Sele 15.39 (7.704 to 25.96) 1420380_at chemokine (C-C motif) ligand 2 Ccl2 14.03 (4.487 to 60.87) 1421228_at chemokine (C-C motif) ligand 7 Ccl7 12.91 (5.766 to 37.1) 1419728_at chemokine (C-X-C motif) ligand 5 Cxcl5 12.76 (6.372 to 20.77)
1433699_at tumor necrosis factor, alpha-induced protein 3 Tnfaip3 11.51 (8.206 to 15.86)
1438148_at gene model 1960, (NCBI) Gm1960 7.572 (3.422 to 14.93) 1419132_at toll-like receptor 2 Tlr2 6.841 (4.335 to 9.101) 1415989_at vascular cell adhesion molecule 1 Vcam1 6.474 (4.739 to 7.946) 1448162_at vascular cell adhesion molecule 1 Vcam1 6.271 (5.523 to 7.878) 1441855_x_at chemokine (C-X-C motif) ligand 1 Cxcl1 6.134 (2.617 to 14.59) 1424067_at intercellular adhesion molecule Icam1 5.442 (3.092 to 9.412)
1424923_at serine (or cysteine) peptidase inhibitor, clade A, member 3G
Serpina3g 5.407 (3.646 to 11.07)
1436003_at vascular cell adhesion molecule 1 Vcam1 5.376 (4.92 to 6.145) 1420697_at solute carrier family 15, member 3 Slc15a3 5.316 (4.142 to 8.184) 1427689_a_at TNFAIP3 interacting protein 1 Tnip1 4.582 (3.627 to 5.967)
1448306_at nuclear factor of kappa light chain gene enhancer in B-cells inhibitor, alpha Nfkbia 4.447 (2.798 to 6.527)
1417065_at early growth response 1 Egr1 4.385 (2.458 to 6.346)
1458299_s_at nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, epsilon Nfkbie 4.122 (2.365 to 7.285)
1450829_at tumor necrosis factor, alpha-induced protein 3 Tnfaip3 4.118 (3.007 to 4.873)
1448914_a_at colony stimulating factor 1 (macrophage) Csf1 4.005 (2.548 to 5.647)
1431843_a_at nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, epsilon Nfkbie 3.959 (2.672 to 5.816)
1451314_a_at vascular cell adhesion molecule 1 Vcam1 3.837 (3.282 to 4.252) 1425155_x_at colony stimulating factor 1 (macrophage) Csf1 3.818 (2.472 to 4.975) 1421392_a_at baculoviral IAP repeat-containing 3 Birc3 3.774 (2.909 to 5.175)
1438160_x_at solute carrier organic anion transporter family, member 4a1 Slco4a1 3.68 (2.675 to 5.886)
1427683_at early growth response 2 Egr2 3.674 (2.35 to 4.82)
1449591_at caspase 11, apoptosis-related cysteine peptidase Casp11 3.654 (2.244 to 6.303)
1417483_at nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, zeta Nfkbiz 3.596 (2.354 to 4.931)
1425154_a_at colony stimulating factor 1 (macrophage) Csf1 3.512 (2.409 to 4.616)
1438855_x_at tumor necrosis factor, alpha-induced protein 2 Tnfaip2 3.478 (2.48 to 5.003)
1427682_a_at early growth response 2 Egr2 3.45 (2.841 to 4.569) 1420549_at guanylate nucleotide binding protein 1 Gbp1 3.338 (2.645 to 4.427) 1460220_a_at colony stimulating factor 1 (macrophage) Csf1 3.335 (2.179 to 4.725) 1447927_at macrophage activation 2 like Mpa2l 3.226 (2.836 to 3.497)
1438157_s_at nuclear factor of kappa light chain gene enhancer in B-cells inhibitor, alpha Nfkbia 3.208 (1.823 to 4.72)
1416273_at tumor necrosis factor, alpha-induced protein 2 Tnfaip2 3.198 (2.095 to 5.142)
1455197_at Rho family GTPase 1 Rnd1 3.186 (2.713 to 4.281) 1421207_at leukemia inhibitory factor Lif 3.104 (1.754 to 4.481) 1438676_at macrophage activation 2 like Mpa2l 3.065 (2.619 to 3.977)
1430752_at RIKEN cDNA C330006D17 gene C330006D17Rik 3.028 (2.349 to 3.69)
1425902_a_at nuclear factor of kappa light polypeptide gene enhancer in B-cells 2, p49/p100 Nfkb2 2.997 (2.041 to 4.286)
1448728_a_at nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, zeta Nfkbiz 2.932 (2.515 to 3.941)
1454903_at nerve growth factor receptor (TNFR superfamily, member 16) Ngfr 2.93 (1.847 to 5.379)
1438498_at zinc finger, MYND domain containing 15 LOC574428 2.889 (2.297 to 3.996)
1436329_at early growth response 3 Egr3 2.864 (1.94 to 4.909)
1418539_a_at protein tyrosine phosphatase, receptor type, E Ptpre 2.857 (2.051 to 3.443)
1418540_a_at protein tyrosine phosphatase, receptor type, E Ptpre 2.847 (2.32 to 3.286)
1452483_a_at CD44 antigen Cd44 2.824 (1.995 to 4.423)
1421008_at radical S-adenosyl methionine domain containing 2 Rsad2 2.823 (2.208 to 3.439)
1419431_at epiregulin Ereg 2.811 (2.537 to 3.242)
1435595_at RIKEN cDNA 1810011O10 gene
1810011O10Rik 2.765 (1.893 to 4.798)
1418718_at chemokine (C-X-C motif) ligand 16 Cxcl16 2.761 (1.977 to 4.019)
1420089_at Nuclear factor of kappa light chain gene enhancer in B-cells inhibitor, alpha (Nfkbia) Nfkbia 2.742 (1.536 to 4.352)
1454976_at Superoxide dismutase 2, mitochondrial, mRNA (cDNA clone IMAGE:3587227) Sod2 2.704 (2.043 to 3.161)
1421009_at radical S-adenosyl methionine domain containing 2 Rsad2 2.7 (1.544 to 4.071)
1453228_at syntaxin 11 Stx11 2.669 (2.432 to 3.195)
1449773_s_at growth arrest and DNA-damage-inducible 45 beta
Gadd45b 2.651 (2.122 to 3.471)
1449195_s_at chemokine (C-X-C motif) ligand 16 Cxcl16 2.647 (1.964 to 3.424)
1427348_at zinc finger CCCH type containing 12A Zc3h12a 2.646 (2.127 to 2.969)
1418675_at oncostatin M receptor Osmr 2.609 (1.782 to 3.61) 1423635_at bone morphogenetic protein 2 Bmp2 2.6 (2.273 to 2.871) 1460251_at Fas (TNF receptor superfamily member) Fas 2.598 (2.176 to 3.21) 1435906_x_at guanylate nucleotide binding protein 2 Gbp2 2.581 (2.52 to 2.63)
1418240_at guanylate nucleotide binding protein 2 Gbp2 2.57 (2.308 to 2.805) 1439221_s_at CD40 antigen Cd40 2.558 (2.277 to 2.816)
1436058_at radical S-adenosyl methionine domain containing 2 Rsad2 2.534 (1.508 to 3.465)
1434376_at CD44 antigen Cd44 2.517 (2.383 to 2.683) 1449473_s_at CD40 antigen Cd40 2.516 (2.113 to 3.414) 1455000_at G protein-coupled receptor 68 Gpr68 2.51 (2.079 to 3.535)
1435644_at RIKEN cDNA G431001E03 gene G431001E03Rik 2.503 (2.402 to 2.67)
1436614_at expressed sequence AI843639 AI843639 2.498 (1.706 to 3.118)
1450004_at thymic stromal lymphopoietin Tslp 2.494 (2.086 to 2.825) 1420499_at GTP cyclohydrolase 1 Gch1 2.484 (2.111 to 2.83) 1427736_a_at chemokine (C-C motif) receptor-like 2 Ccrl2 2.472 (2.272 to 2.597)
1442018_at expressed sequence AI426953 AI426953 2.455 (1.858 to 3.478)
1417488_at fos-like antigen 1 Fosl1 2.428 (2.077 to 3.181)
1425452_s_at protein tyrosine phosphatase, receptor type, J /// expressed sequence AW125753
Ptprj /// AW125753 2.404 (2.208 to 2.571)
1423760_at CD44 antigen Cd44 2.391 (2.148 to 2.806) 1435888_at epidermal growth factor receptor Egfr 2.381 (2.034 to 3.043)
1450173_at receptor (TNFRSF)-interacting serine-threonine kinase 2 Ripk2 2.365 (2.174 to 2.748)
1439774_at paired related homeobox 1 Prrx1 2.355 (2.037 to 2.683) 1451458_at transmembrane protein 2 Tmem2 2.33 (1.792 to 3.542) 1418854_at baculoviral IAP repeat-containing 2 Birc2 2.321 (2.05 to 2.856) 1460415_a_at CD40 antigen Cd40 2.271 (1.736 to 3.002)
1444003_at lung-inducible neuralized-related C3HC4 RING domain protein
MGI:2429944 2.265 (1.852 to 2.915)
1428420_a_at RIKEN cDNA 1200009I06 gene 1200009I06Rik 2.244 (1.869 to 2.967)
1420710_at reticuloendotheliosis oncogene Rel 2.208 (1.653 to 3.26) 1419647_a_at immediate early response 3 Ier3 2.198 (2.152 to 2.293)
1452339_at a disintegrin-like and metallopeptidase with thrombospondin type 1 motif, 7
Adamts7 2.19 (1.577 to 3.606)
1444531_at Superoxide dismutase 2, mitochondrial, mRNA (cDNA clone IMAGE:3587227) Sod2 2.18 (1.69 to 2.638)
1418872_at ATP-binding cassette, sub-family B (MDR/TAP), member 1B Abcb1b 2.178 (1.953 to 2.354)
1418936_at v-maf musculoaponeurotic fibrosarcoma oncogene family, protein F (avian) Maff 2.174 (1.838 to 2.66)
1417487_at fos-like antigen 1 Fosl1 2.173 (1.915 to 2.601)
1435596_at RIKEN cDNA A530088I07 gene A530088I07Rik 2.155 (1.69 to 2.648)
1438097_at RAB20, member RAS oncogene family Rab20 2.151 (1.816 to 2.415) 1434600_at tight junction protein 2 Tjp2 2.119 (2.023 to 2.174)
1438673_at solute carrier family 4, sodium bicarbonate cotransporter, member 7 Slc4a7 2.119 (1.923 to 2.54)
1419212_at icos ligand Icosl 2.118 (1.45 to 3.047)
1457780_at PREDICTED: syntaxin 11 [Mus musculus], mRNA sequence Stx11 2.111 (1.633 to 2.563)
1452078_a_at solute carrier family 11 (proton-coupled divalent metal ion transporters), member 2 Slc11a2 2.108 (1.657 to 2.786)
1429692_s_at GTP cyclohydrolase 1 Gch1 2.098 (1.97 to 2.275)
1457528_at solute carrier family 4, sodium bicarbonate cotransporter, member 7 Slc4a7 2.08 (1.588 to 3.202)
1423025_a_at schwannomin interacting protein 1 Schip1 2.067 (1.607 to 2.694)
1423103_at regulatory factor X, 5 (influences HLA class II expression) Rfx5 2.053 (1.678 to 2.943)
1418674_at oncostatin M receptor Osmr 2.053 (1.462 to 2.723) 1427005_at polo-like kinase 2 (Drosophila) Plk2 2.048 (1.493 to 2.72)
1450350_a_at Jun dimerization protein 2 MGI:1932093 2.03 (1.533 to 2.781)
1457824_at Phospholipid scramblase 1, mRNA (cDNA clone MGC:5827 IMAGE:3491904) Plscr1 2.026 (1.715 to 2.217)
1417292_at interferon gamma inducible protein 47 Ifi47 2.016 (1.771 to 2.382) 1418392_a_at guanylate nucleotide binding protein 4 Gbp4 2.012 (1.565 to 2.771) 1418186_at glutathione S-transferase, theta 1 Gstt1 0.466 (0.339 to 0.65) 1434089_at synaptopodin Synpo 0.463 (0.409 to 0.528)
1441687_at Wingless-related MMTV integration site 4 (Wnt4), mRNA Wnt4 0.457 (0.343 to 0.762)
1417234_at matrix metallopeptidase 11 Mmp11 0.444 (0.346 to 0.532)
1427149_at pleckstrin homology domain containing, family A member 6 Plekha6 0.443 (0.339 to 0.563)
1447862_x_at thrombospondin 2 Thbs2 0.438 (0.303 to 0.573) 1423946_at PDZ and LIM domain 2 Pdlim2 0.428 (0.302 to 0.61)
1431079_at C1q and tumor necrosis factor related protein 2 C1qtnf2 0.426 (0.28 to 0.66)
1424007_at growth differentiation factor 10 Gdf10 0.417 (0.366 to 0.475) 1455422_x_at septin 4 Sept4 0.417 (0.362 to 0.496) 1454880_s_at Bcl2 modifying factor Bmf 0.416 (0.384 to 0.445) 1434326_x_at coronin, actin binding protein, 2B Coro2b 0.414 (0.278 to 0.637)
1429726_at solute carrier family 16 (monocarboxylic acid transporters), member 9 Slc16a9 0.411 (0.372 to 0.442)
1438211_s_at D site albumin promoter binding protein Dbp 0.387 (0.277 to 0.475) 1448700_at G0/G1 switch gene 2 G0s2 0.382 (0.331 to 0.505) 1418890_a_at RAB3D, member RAS oncogene family Rab3d 0.38 (0.249 to 0.486) 1454708_at actin-binding LIM protein 1 Ablim1 0.376 (0.271 to 0.457) 1423967_at paralemmin Palm 0.369 (0.272 to 0.616) 1436996_x_at P lysozyme structural Lzp-s 0.367 (0.21 to 0.507) 1426418_at atonal homolog 8 (Drosophila) Atoh8 0.366 (0.297 to 0.474)
1423753_at BMP and activin membrane-bound inhibitor, homolog (Xenopus laevis) Bambi 0.365 (0.328 to 0.406)
1417580_s_at selenium binding protein 1 Selenbp1 0.326 (0.277 to 0.356)
1450699_at selenium binding protein 1 Selenbp1 0.315 (0.291 to 0.331)
1418804_at succinate receptor 1 Sucnr1 0.305 (0.221 to 0.491)
1429175_at RIKEN cDNA 2810417M05 gene
2810417M05Rik 0.301 (0.198 to 0.418)
1450782_at wingless-related MMTV integration site 4 Wnt4 0.269 (0.201 to 0.362)
