Supplementary Information
Materials and methods
Generation of a conditional allele of the Mcl-1 gene
To generate a conditional (floxed) allele of the Mcl-1 gene, a targeting vector as depicted in
supplemental Figure S1A was constructed using a genomic DNA fragment harboring the
Mcl-1 gene locus isolated from a 129/Svj mouse genomic library. Within this targeting vector
a floxed Neo cassette and a third loxP site were inserted into intron 2 and intron 1 of the
Mcl-1 gene, respectively. A pgk promoter-driven TK cassette was included as indicated for
negative selection. This targeting vector was electroporated into R1 embryonic stem (ES)
cells and three clones that had undergone homologous recombination at the Mcl-1 locus were
selected via genomic Southern analysis using 5’ and 3’ probes as indicated in Figure S1A.
One ES clone harboring the “targeted allele” was further microinjected into C57BL/6
blastocysts to generate chimeric mice. Male chimeric mice were backcrossed with C57BL/6
females to generate heterozygous mice. To generate mice carrying the “floxed allele” (f)
(Figure S1A), heterozygous mice with a targeted allele were mated with EIIa-CRE transgenic
mice [1] (originally in FVB/N background but backcrossed to C57BL/6 for more than 7
generations, a gift provided by Ying-Hue Lee at IMB, Academia Sinica) to remove the Neo
cassette in the targeted allele. Mcl-1f/f mice were then generated by heterozygous crosses and
verified by Southern blotting as well as PCR analysis using genomic DNA isolated from the
mouse tail. The “floxed allele”, upon Cre-mediated deletion, would become the “deleted
allele”, which is a null allele, because homozygous mutants with this allele (Mcl-1-/-)
specifically generated in hepatocytes did not produce any full-length Mcl-1 proteins.
Routine genotyping in this study was performed by PCR using primers as specified below
(Numbers in bracket indicate the predicted size of the amplified DNA fragment): For the Cre
transgene: sense, 5’-TGCCACGACCAAGTGACAGC-3’, anti-sense, 5’-CC
TTAGCGCCGTAAATCAATCG-3’ [519 bp]; Mcl-1 wt or floxed allele, sense (P6),
5’-TTCTTGTA AGGACGAAACGGGACT-3, anti-sense (P7),
5’-TTTCTCTGGACCTACCACC TACAA-3’ [wt, 218 bp; f, 318 bp]; P53 WT allele, sense,
5’-GGGAGGGACAAAAGTTCGAGG-3’, anti-sense, 5’-CTGTCTTCCAGAT
ACTCGGGATAC-3’ [666 bp]; P53 KO allele, sense, 5’-TCTATCGCCTTCTTGACG AG-3’,
anti-sense, same as that for the p53 WT allele [257 bp].
Histological analysis and immunofluorescence microscopy
Liver specimens were fixed with 4% PBS-buffered paraformaldehyde, dehydrated with
graded ethanol and embedded in paraffin. Four-micrometer-thick tissue sections were
deparaffinized in Histoclear II, rehydrated and subjected to hematoxylin and eosin (H&E) or
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Sirius red staining according to standard protocols. For some experiments, liver sections were
stained with FITC-labeled goat anti-albumin antibody (Bethyl Laboratories, Montgomery, TX)
and specific signals were visualized by confocal fluorescence microscopy (Zeiss type
LSM-meta 510, Berlin, Germany).
Immunohistochemistry
Immunohistochemistry (IHC) was performed essentially as previously described [2], using
antibodies specific to the following proteins: Mcl-1 [3], β-catenin (BD Transduction
Laboratories, San Jose, CA), PCNA (Millipore, Billerica, MA), phospho-histone H3 (pHH3;
BD Transduction Laboratories) or caspase-3 (Cell Signaling, Danvers, MA). Quantifications
of all these IHC were determined by counting cells in at least ten 400x high-power fields in
the sections of the same liver.
In vivo BrdU labeling
Mice were i.p. injected with BrdU (100 mg/kg; Sigma-Aldrich, St. Louis, MO) 2 h before
they were sacrificed for analysis. Immunohistochemical staining for BrdU was performed in
liver sections using a Zymed BrdU staining kit (Zymed, S. San Francisco, CA) according to
the manufacturers’ protocol.
Isolation of hepatocytes and determination of DNA contents
Liver cells were isolated by the collagenase perfusion method essentially as previously
described [4, 5]. To determine the DNA contents of hepatocytes, isolated cells were washed
with PBS and fixed with 70% ethanol at 4℃. After overnight fixation, cell pellets were
resuspended in a solution containing propidium iodide (20ug/ml), RNase A (200U/ml) and
TritonX-100 (0.1%) for 30 min and then subjected to flow cytometric analysis.
Determination of serum interleukin-6 (IL-6) and tumor necrosis factor (TNF- levels
Serum IL-6 and TNF- levels were measured using the DuoSet ELISA Development kit
(R&D Systems) according to the manufacturers’ protocols.
Western blotting Analysis
Whole liver or hepatocytes isolated from mice to be analyzed were lysed in the RIPA
buffer (10mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 0.1%
sodium dodecyl sulfate, and 1% deoxycholate) containing 1mM PMSF, aprotinin (1 ug/ml)
and leupeptin (1 ug/ml). Fifty ug of protein lysates were resolved by 12% SDS-PAGE,
transferred to polyvinylidene difluoride membranes (Millipore) and probed with antibodies as
specified in each figure. Antibodies used include those specific to mouse Mcl-1 [3], Bcl-xL,
Bax, (all from Santa Cruz Biotechnology), Bim (ProSci), t-Bid (Millipore), Bak (Upstate),
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tubulin (Sigma-Aldrich) and p53 (BD Transduction Laboratories). After incubation with an
appropriate horseradish peroxidase-conjugated secondary antibody, specific signals were
revealed by an ECL Western blot system (Amersham Pharmacia Biotech).
TUNEL staining
For detection of apoptotic cells in the liver sections, the terminal deoxynucleotidyl
transferase–mediated dUTP nick end-labeling (TUNEL) assay was carried out essentially as
previously described [6]. After TUNEL staining, liver sections were counterstained with
hematoxylin. The percentage of TUNEL-positive cells was determined by counting cells in at
least ten 400X fields in the sections from the same liver.
Real-Time quantitative PCR
Total RNA from the liver of mice at 4-8 weeks of age was isolated using the Trizol reagent
(Invitrogen,Carlsbad, CA), and reverse-transcribed into cDNA using oligo (dT) primers and
the Moloney Murine Leukemia virus reverse transcriptase (Invitrogen, Carlsbad, CA). The
expression levels of gene of interests and GAPDH mRNA were analyzed by real-time
quantitative RT-PCR on the LightCycler 480 System according to the manufacturer’s
protocol (Roche Applied Science, Indianapolis, IN). The LightCycler 480 software version
1.5 was used for data analysis. All mRNA levels were normalized to that of GAPDH by
subtracting the crossing point (CP) value of GAPDH from that of the test gene (ΔCP = CPTest –
CPGAPDH). The sequences of the primers used in this assay are as follows: AFP, sense,
5’-TCGTATTCCAACAGGAGG-3’ and anti-sense, 5’-AGGCTTTTGCTTCACCAG-3’;
Cdkn1a, sense, 5’-CCTGGTGATGTCCGACCTG-3’ and anti-sense,
5’-CCATGAGCGCATCGCAATC-3’; Ccng1, sense,
5’-TGGACAGATTCTTGTCTAAAATGAAG-3’ and anti-sense,
5’-CAGTGGGACATTCCTTTCCTC-3’; Rrm2b, sense,
5’-TGCCTGATGTTTCAGTACTTGG-3’ and anti-sense,
5’-ACAGGCAAGGCTTCTGTTAAA-3’; Wig1, sense,
5’-AAGCCCAGGCACACTATCAG-3’ and anti-sense, 5’-CTACAGGCTCTGCCACCAC-3’;
GAPDH, sense, 5’- GGTGAAGCAGGCATCTGAGG-3’ and antisense, 5’AGGGAGATGCTCAGTGTTGG-3’.
Results
Increased sensitivity of DKO mice to CD95-mediated apoptosis
We also examined the sensitivity of DKO mice to CD95-mediated apoptosis, because
hepatocytes constitutively express CD95 [7]. For this experiment, mice at the age of 14-20
weeks were i.p. injected with the agonistic CD95 antibody Jo2. Three hours after Jo2
treatment, mice were sacrificed and analyzed. As shown in Figure S5, the serum levels of both
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AST and ALT were markedly increased in DKO mice compared to control, p53-/- or
Alb-Mcl-1-/- mice. The Jo2 treatment also significantly increased the serum levels of two
inflammatory cytokines IL-6 and TNF-in DKO mice compared to mice with three other
genotypes (Figure S5). Of note, the basal levels of these two inflammatory cytokines in
untreated DKO mice were as low as those in untreated control, p53-/- or Alb-Mcl-1-/- mice
(Figure S5), suggesting that only severe liver injury following Jo2 treatment, but not the basal
level of spontaneous liver damage in DKO mice led to liver inflammation.
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