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Supplemental Research Data
Supplemental Experimental Procedures
Generation of Crif1 knockout mice. To disrupt Crif1, a targeting vector was designed in
which the 2.2 Kb ClaI/EcoRV fragment (Figure S1A) was deleted and replaced with the
neomycin phosphotransferase (neo) gene, under the control of the phosphoglycerate kinase
promoter (pgk). Homologous recombination at the Crif1 locus resulted in the deletion of exon 1
(Figure S1A). Three E14K ES cells obtained by homologous recombination were used to
generate chimeric mice after injection into C57BL/6 blastocysts. Subsequent breedings were
done with C57BL/6 mice to generate congenic mice and with FVB/N to test the effects of the
genetic background. The genotype analyses were carried out by PCR using the following
primers: wildtype forward 5’-ACAGAGAGCCGCAGGAGATAG-3’; mutant forward 5’CGAAGGGGCCACCAAAGA ACG-3’ and common 5’-GCCAACGCACGACACTTTT-3’.
Generation of Crif1 conditional mice. A second targeting vector was designed to conditionally
delete exon 2, which was flanked by two loxp sites (Figure S3A). The vector was introduced by
electroporation into E14K ES cells, and drug-resistant colonies were screened for homologous
recombination. Five of 386 clones were determined to be positive by Southern blotting, using a
5’ external probe (Figure S3B). Hybridization with the 5’ external probe revealed a 6.3kb Xba1
fragment for the wild-type allele and a 7.7kb fragment for the floxed allele (Figure S3B). Two
ES clones were used to generate chimeric mice and to obtain germline transmission. The
genotype analyses of the animals, embryos, and cultured embryonic fibroblasts were carried out
by PCR using the following primers: F_TCAGCTAGGGTGGGACAGA, and R_GGGCTGGT
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GAAATGTGTTG. These primers amplified a 171bp band from the wild type allele and a 205bp
band from the targeted allele, due to the insertion of the loxp site.
In situ hybridization. Details of the RNA in situ hybridizations on whole mount embryos have
been described (Koo et al., 2005). Antisense DIG-labeled (digoxigenin) riboprobes were
generated from pGEM-T vectors (Promega) containing amplified cDNA fragments (about
500~700 bp). Staining patterns were confirmed by comparisons with previously published data,
except for crif1.
RT-PCR analysis. Total RNA from blastocyst cultures and MEFs was extracted using
an RNeasy Micro kit (Qiagen) and Trizol reagent (Life Technologies), respectively,
according to the manufacturers’ instructions. Complementary DNA synthesis was
performed according to the manufacturer’s instructions (Omniscript kit; Qiagen). Realtime RT-PCR reactions with SybrGreen quantification were set up with 1/25 of each
cDNA preparation in a Roche LightCycler. Relative expression levels and statistical
significance were calculated based on an Oct4 standard, using the LightCycler software.
All amplicons (100~200 bp) showed efficient amplification, which allowed us to equate
one threshold cycle difference. Primer information is provided in the supplementary
material.
Western blot analysis and co-immunoprecipitation assay. Cultured cells were
resuspended in IP buffer [50 mM HEPES/NaOH (pH 7.5), 3 mM EDTA, 3 mM CaCl2,
80 mM NaCl, 1% Triton X-100, 5 mM DTT]. Generally, the protein in the supernatants
(25~40 g) was separated by size, blotted with primary and secondary Abs and
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visualized with ECL plus (Amersham Biosciences). The primary Abs used were as
follows: anti-c-myc, anti-HA, anti-Flag, anti-STAT1, anti-Actin (Santa Cruz
Biotechnology), anti-STAT3, anti-phospho STAT3 (Cell Signaling), and anti-STAT5a
(Upstate Biotechnology). Immunoprecipitation was performed as previously described
(Koo et al., 2005).
Preparation of the anti-Crif1 polyclonal antibody. To produce the anti-Crif1 antibody,
synthetic peptides corresponding to mouse Crif1 amino acids 23-38 and 138-153 were
coupled through the carboxyl-terminal cysteine to KLH, and the conjugates were used
to immunize New Zealand white rabbits. Affinity purification of the peptide-specific
IgG was accomplished by coupling the peptides to Affi-Gel (Bio-Rad).
In-situ hybridization probe information.
crif1: F_ ATGGCGGCGCTCGCAAT, R_ CCAGACACTGCTGAGTCC
oct4: F_ TTCAGACTTCGCCTCCTCAC, R_ ACTCCACCTCACACGGTTCT
nanog: F_ CTCTCCTCGCCCTTCCTC, R_ AGGCTTGTGGGGTGCTAAA
RT-PCR primer information.
crif1: F_TATCTCCTGCGGCTCTCTGT, R_ CTTCTGCTTTCGCCAGTTTT
oct4: F_ GGCGTTCTCTTTGGAAAGGTGTTC, R_ CTCGAACCACATCCTTCTCT
myc: F_ GTCTCCACTCACCAGCACAA, R_ CGTCGTTTCCTCAATAAGTCC
socs3: F_ CCTGCGCCTCAAGACCTTCA , R_ CGGCTCAGTACCAGCGGAAT
c-fos: F_ CTGCGTACTTGCTTCTCCTAATAC, R_ GAGTGTTCACATTTGGGATCTT
junB: F_GCTGGAGAAAGCGGAGATACT, R_ AAAGCCCTCCTGCTCCTC
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Id1: F_ CACTCTGTTCTCAGCCTCCTC, R_ CTTGCTCACTTTGCGGTTCT
Id3: F_ CTGCTACGAGGCGGTGTG, R_ TGTCTGGATCGGGAGATG
sox2: F_ ACAGCTACGCGCACATGAA, R_ CTGGAGTGGGAGGAAGAGG
cd44: F_ TCCAACACCTCCCACTATGAC, R_ TACTCGCCCTTCTTGCTGTAG
irf1:F_GCACTGTCACCGTGTGTCGTCAGCAG,
R_TGTCCCCTCGAGGGCTGTCAATCTCTG
Chromatin immunoprecipitation primer information.
myc: F_ AAAAATAGAGAGAGGTGGGGAAG, R_TGGAATTACTACAGCGAGTCAGAA
socs3: F_ CAGGCGAGTGTAGAGTCAGAGTT, R_ CACAGCCTTTCAGTGCAGAGTAG
c-fos: F_ GCGTACTTGCTTCTCCTAATACCA, R_ GAGTGTTCACATTTGGGATCTT
Supplementary Figure legends
Supplementary Figure 1. Interaction of Crif1 with STAT3
(A) Yeast two-hybrid assays testing the interaction between Crif1 and STAT3. (B) Schematic
representation of the Crif1 deletion mutants. NTD, N-terminal domain; NLS, nuclear
localization signal; CCD, coiled-coil domain. (C) Schematic representation of the STAT3
deletion mutants. (D, E) Flag-tagged STAT3 deletion mutants (D) and Myc-tagged STAT3
deletion mutants (E) were co-transfected with HA-tagged full length Crif1, and then
immunoprecipitated with an anti-HA Ab. Total cell extracts and immunoprecipitates were
blotted with anti-HA, anti-Flag and anti-Myc Abs. (F) V5-tagged STAT3 and V5-tagged
STAT3Y705F plasmids were each co-electroporated with HA-tagged Crif1 into the
MEFs, which were then stimulated with OSM for 20 min. Anti-HA immunoprecipitates
from the whole cell extracts were subjected to western blot analyses with anti-HA and
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anti-V5 Abs.
Supplementary Figure 2. Targeted disruption of the murine Crif1 locus leads to embryonic
lethality
(A) The targeting construct is depicted below the endogenous mouse Crif1 locus, showing two
exons (black squares). The flanking genomic probe used for Southern hybridization is indicated
with a black bar. B, BamHI; S, SpeI; C, ClaI; Ev, EcoRV; neo, neomycin phosphotransferase;
DTA, Diphtheria toxin A. (B) Southern blot analysis of BamHI-digested genomic DNA from
targeted ES cell clones, probed with the flanking genomic probe shown in A. An 8 kb fragment
is detected from the untargeted locus, while the 7 kb fragment indicates the targeted locus. (C)
PCR analysis of representative genomic tail DNA from Crif1 heterozygous intercrosses,
showing the wild-type and targeted alleles. (D) Genotype analysis of the progeny from Crif1
heterozygous intercrosses.
Supplementary Figure 3. Crif1 expression in early development
(A-C) Whole-mount in-situ hybridization in wild-type embryos. Crif1 mRNA is visible in the
ICM of the blastocyst (A), in an identical manner as Oct4 and Nanog (B and data not shown),
and in the embryonic and extraembryonic tissues of the E7.5 embryo (C). ch, chorion; al,
allantois; am, amnion. (D) Crif1 expression in the early developmental stages of embryos. Total
RNA was isolated from the embryos at different stages and was analyzed with specific primers
for Crif1. 1, unfertilized eggs; 2, 1 cell stage; 3, 2 cell stage; 4, 4 cell stage; 5, 8 cell stage; 6,
morula stage; 7, blastocyst stage. (E and F) Whole-mount in situ hybridization of 68 blastocysts
from 7 heterozygote intercrosses. Blastocysts were divided into three groups for the in situ
hybridization of Crif1 (E) and Oct4 (F) and the genotyping experiment.
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Supplementary Figure 4. Decreased expression of STAT3 target genes in the colony from
Crif1-/- blastocysts.
RT-PCR analyses of STAT3 target genes (Myc, Socs3, c-Fos, and JunB) and unrelated genes
(Oct4, Id1, Id3, and Sox2). Crif1+/+ (+/+), Crif1+/- (+/-) and Crif1-/- (-/-) blastocysts were
individually cultured in LIF-containing ES medium for 3 days. One-third of an individual
colony was used for genotyping by genomic PCR, and the rest of the colony with the same
genotype was pooled. RNA was extracted from each pooled colony lysate and analyzed by
semiquantitative RT-PCR.
Supplementary Figure 5. Conditional disruption of the Crif1 gene using the Cre-loxp
system
(A) Schematic representations of the targeting vector, the wild-type Crif1 locus, and the
recombined locus. Exon 2 of Crif1 was flanked by two identically oriented loxp sites (triangles).
The flanking genomic probe used for Southern hybridization is indicated with a black bar. Xb,
XbaI; H, HindIII; B, BamHI; S, SpeI; C, ClaI; Ev, EcoRV; neo, FRT sites flanking neomycin
phosphotransferase; DTA, Diphtheria toxin A. (B) Southern analysis of XbaI-digested genomic
DNA from targeted ES cell clones, probed with the flanking genomic probe shown in (A). A 6.3
kb fragment is detected from the untargeted locus, while the 7.7 kb fragment indicates the
floxed locus. (C) PCR typing analysis of DNA from Crif1flox/+ (flox/+) and Crif1flox/- (flox/-)
MEFs, untreated (-) or infected with a Cre retrovirus (+). (D) Protein lysates from Crif1+/
(+/) and Crif1-/ (-/) MEFs were subjected to a western blot analysis with an anti-Crif1 Ab.
Supplementary Figure 6. Crif1 is dispensable for the phosphorylation, dimerization and
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nuclear translocation of STAT3
(A) Normal phosphorylation of STAT3. Crif1+/ (+/) and Crif1-/ (-/) MEFs generated by the
Cre-loxp system were stimulated with OSM (5 ng/ml) for the indicated times, and
phosphorylated STAT3 protein was detected by an anti-phospho-STAT3 Ab. (B) Dimerization of
STAT3. Crif1+/ (+/) and Crif1-/ (-/) MEFs were co-electroporated with flag-tagged STAT3
and V5-tagged STAT3, and were stimulated with OSM (5 ng/ml) for 20 min. Lysates were
immunoprecipitated with an anti-Flag Ab, followed by western blotting with anti-Flag
and anti-V5 Abs. (C) Nuclear translocation of STAT3. Crif1+/ (+/) and Crif1-/ (-/) MEFs
were stimulated with OSM (5 ng/ml) for the indicated times, and immunocytochemistry was
performed with an anti-STAT3 Ab. (D) Crif1+/ (+/) and Crif1-/ (-/) MEFs were stimulated
with OSM (5 ng/ml) for the indicated times, and the expression levels of endogenous STAT3
were detected by western blotting with an anti-STAT3 Ab.
Supplementary Figure 7. Impaired expression of STAT3 target genes
(A-D) Crif1+/ and Crif1-/ MEFs were treated with OSM (5 ng/ml) for the indicated times, and
the indicated gene expression was analyzed by real-time RT-PCR (A-C) and RT-PCR (D).
*P<0.0674, **P<0.01. (E) Crif1+/ and Crif1-/ MEFs were treated with INF (20 ng/ml) for the
indicated times, and the indicated gene expression was analyzed by RT-PCR. The results are
representative of three independent experiments.
Supplementary Figure 8. Defective DNA binding activity of STAT3 in Crif1-/ MEFs
(A) Chromatin immunoprecipitation (ChIP). Soluble chromatin was prepared from
Crif1+/ and Crif1-/ MEFs treated with OSM (5 ng/ml) for the indicated times and was
immunoprecipitated with the anti-STAT3 antibody. The final DNA extracts were
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amplified using pairs of primers that cover the STAT3 binding sites of Myc, Socs3, and
c-Fos. The results are representative of three independent experiments. (B) Abolishment
of DNA binding by STAT3-C. The Crif1+/ and Crif1-/ MEFs, immortalized using
SV40 large T antigen and stably overexpressing STAT-C, were subjected to chromatin
immunoprecipitation with the anti-STAT3 Ab. DNA amplification was performed as in
(A). The results are representative of four independent experiments. (C) The
recruitment of HA-Crif1 to the STAT3-responsive region. HA-Crif1 NIH3T3 cells were
stimulated with OSM (5 ng/ml) for the indicated times. Chromatin complexes were
immunoprecipitated with anti-HA or anti-STAT3 Abs. DNA amplification was
performed as in (A). The results are representative of three independent experiments.
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