Supporting Online Material for
CD24 and Siglec-10 Selectively Repress Tissue Damage–Induced
Guo-Yun Chen, Jie Tang, Pan Zheng,* Yang Liu*
*To whom correspondence should be addressed. E-mail: firstname.lastname@example.org (Y.L.),
Published 5 March 2009 on Science Express
This PDF file includes:
Materials and Methods
Figs. S1 to S4
Materials and Methods
Reagents Recombinant proteins consisting of human IgG Fc and extracellular
domains of SIglec 5, 7, 10 and 11 were purchased from R&D Systems. Horse-
radish perioxidase conjugated anti-mouse, or anti-rabbit secondary-step
reagents, as well as anti-p65 and anti-sp1 were purchased from Santa Cruz
Biotechnology. Anti-FLAG M2 affinity gel, anti-FLAG mAb, acetaminophen
(AAP) and lipopolysaccharide (LPS, from E. coli 055:B5) were purchased from
Sigma (St Louis, MO). The composition CD24Fc have been described 1, the
product is obtained from OncoImmune, Inc. (Columbus, OH). Human HSP70,
HSP90 and anti-mouse Hsp70, Hsp90 antibodies were purchased from
Biovision, Inc. (Mountain View, CA). The anti-HMGB-1 antibodies 3E8 and 3B1
were described in supplemental information.
cDNAs encoding either full-length or specifically truncated human HMGB-
1 and N-FLAG-tagged WT or mutant (119R>A) Siglec10 were cloned into
expression vector pCMV-Tag 2B (Sigma). All constructs were verified by DNA
sequencing. For purification of FLAG-tagged HMGB-1, the full-length HMGB-1
expression vector was transfected into TSA cells, the lysates were used as
source to purify recombinant HMGB-1 according to a reported procedure 2.
Experimental animals Mice with targeted mutations of CD24 and Siglecg were
produced from ES cells of C57BL/6 origin as have been described 3, 4. Age- and
sex-matched wild type C57BL/6 mice were used as controls. All mice were used
at 6-8 weeks of age. All procedures involving mice have been approved by the
University of Michigan Animal Care and Use Committee.
Mouse pathology For ALT measurements, blood was collected at given time
points. Serum was isolated by centrifugation of clotted blood at 12,000 x g for 10
min at room temperature and then sent to Animal Diagnostic Laboratory of
Animal Research Facility, University of Michigan (Ann Arbor, USA) for
determining ALT activity. For histology, liver was removed and immediately fixed
in 4% formaldehyde-PBS solution, embedded in paraffin, sectioned at 5 μm, and
stained with hematoxylin and eosin. Serum cytokines were determined using
mouse cytokine bead array designed for inflammatory cytokines (Cat. No
552364, BD Biosciences).
Flow cytometric analysis for SIglec10 ligands Spleen cells from WT or
CD24-/- mice were washed in buffer A (150 mM NaCl, 3 mM MnCl2, 1 mM CaCl2,
1 mM MgCl2, 25mM Tris, pH 7.6, 2% BSA), and incubated for 1 hour at 37 °C
with 1 μg of Siglec-10-Fc or Fc control. The bound receptors were detected with
PE conjugated anti-human IgG-Fc and analyzed on a BD LSII.
Immunoprecipitation and immunoblotting Cell lysates were prepared in the
buffer B (1 % Triton X-100, 150 mM NaCl, 3 mM MnCl2, 1 mM CaCl2, 1 mM
MgCl2, 25 mM Tris, pH 7.6) and protease inhibitors (1 μg/ml leupeptin, 1 μg/ml
aprotinin and 1 mM phenylmethylsulfonyl fluoride). Samples were pre-cleared
with 60 μl of protein A-conjugated agarose beads (Upstate, Lake Placid, NY) for
2 h at 4°C or 37°C with rotation, and then incubated with corresponding
antibodies (anti-CD24 mAbs M1/69 and 20C9, 10 μg/ml; anti-HMGB-1, 2 μg/ml;
anti-HSP70 and HSP90 antibodies, 3 μg/ml). The beads were washed four
times with buffer B and re-suspended in SDS sample buffer for Western blot
analyses with given antibodies (0.5 μg/ml). The anti-Siglec-G antisera were
used at 1:100 dilution.
Mass spectrometry After gel concentration, the protein samples were
submitted to Taplin Spectrometry Facility at Harvard Medical School for high
Statistics The differences in cytokine proteins and ALT activities were
analyzed by Student’s t test. The differences in survival rates were analyzed by
Kaplan-Meier survival analysis with log-rank test.
2. Characterization of HMGB-1 antibodies used for the study. HMGB-1 is highly
conserved (98% identity between mouse and human). In order to break immune
tolerance, we introduced a universal T cell epitope from a mycobacterium tuberculosis
Ag 5 into C-terminus of HMGB-1 and the resulting recombinant protein was used as an
antigen for immunization. With the help of the T cell epitope and autoimmune NZB/W
mice, we were able to obtain a panel of mouse anti-HMGB-1 antibodies that cross-react
with mouse and human HMGB-1. Two of them, 3E8 and 3B1 were used in this study.
As shown in Fig. S1a, both antibodies react with recombinant HMGB-1 in Western blot.
In pilot studies, we have found 3E8 to be a more efficient in immunoprecipitation and
Western blot than 3B1 (data not shown). Moreover, 3B1 completely blocked production
of TNFα by DC after stimulation of recombinant HMGB-1 (Fig S1b).
Fig. S1. Characterization of anti-HMGB-1 mAbs used in the study. a.
Immunoblot showing the interaction of 3E8 and 3B1 with recombinant HMGB-1
obtained from R&D system. Note that while 3E8 also binds to a truncated
HMGB-1 in the preparation, 3B1 only recognize the full length form from the
same preparation. Surface Plasmon Resonance (SPR) using the BIACORE3000
revealed that Kd for 3B1-HMGB-1 interaction is 7.8 nM, while that for 3E8-
HMGB-1 interaction is 1.3 nM. b. Inhibition of TNFα production from CD24-/-
dendritic cells, stimulated with HMGB-1 (20 μg/ml) in the presence of given
amounts of 3B1 or mouse IgG control. Supernatants were harvested after 6
hours of culture and measured by cytokine beads array.
500 500500 500
2500 25002500 2500
0000 30 303030 60 6060 60120 120120120 300 300300300 600600 600600
3B1 3B13B1 3B1mouse IgGmouse IgGmouse IgG mouse IgG
TNF (pg/ml) TNF (pg/ml)
Table S1. Confirmation of CD24-HMGB-1 interaction by mass-spectrometry.
The lysates from WT and CD24-deficient hosts were incubated with anti-CD24
mAbs (a mixture of 20C9 and M1/69 and precipitated with protein G beads. The
precipitates were incubated with the EDTA to release cation-dependent binding.
The eluted proteins were subject to trysinization followed by mass-spectrometry
analysis. The data shown are peptides identified from WT spleen cells, and no
HMGB-1 peptides were identified from the immunoprecipitates of the CD24-/-
30-42 HPDASVNFSEFSK 30-42 HPDASVNFSEFSK
Position PositionSequence Sequence
76-85 TYIPPKGETK 76-85 TYIPPKGETK
114-126 GEHPGLSIGDVAK 114-126 GEHPGLSIGDVAK
29-42 KHPDASVNFSEFSK 29-42 KHPDASVNFSEFSK
112-126 IKGEHPGLSIGDVAK 112-126 IKGEHPGLSIGDVAK
128-145 LGEMWNNTAADDKQPYEK 128-145 LGEMWNNTAADDKQPYEK
57-64 GKFEDMAK 57-64 GKFEDMAK
127-145 KLGEMWNNTAADDKQPYEK 127-145 KLGEMWNNTAADDKQPYEK
154-162 YEKDIAAYR 154-162 YEKDIAAYR
Peptide matchesPeptide matches
A box A box
B box +Acidic tailB box +Acidic tail
IP: CD24 Fc
IP: CD24 Fc
Supplemental Fig. S2. CD24 does not bind to inhibitory Box A of HMGB-1.
cDNA encoding FLAG-tagged full-length (F), inhibitory Box A (A) or Box B plus
acidic tail (BC) were transfected into COS7 cells. The cells were lysed and
precipitated with recombinant CD24Fc. The precipitates were blotted with either
anti-FLAG or anti-IgG Fc. The relative amounts of truncated proteins expressed
were measured by anti-FLAG. The positions of the truncated products were
diagrammed on the top.
PE-anti-mouse IgGPE-anti-mouse IgG PE-anti-mouse IgG
Cell number (max%)
Cell number (max%)
Cell number (max%)
Fig. S3. Characterization of anti-Siglec-G antisera. Siglecg-/- mice
were immunized with WT spleen cells (approximately 107/mouse/injection)
that have been stimulated with LPS (10 μg/ml) for 24 hours. After three
immunizations, the sera were collected. (A) Specific binding to WT but not
Siglecg-/- spleen cells. Spleen cells were stained with 1:100 dilution of the
mouse anti-serum, and the IgG bound to the cells were determined by
phycoerythorin-conjugated goat-anti-mouse IgG-Fc. The size of the
positive subset roughly matches what was revealed by the GFP markers
(3). (B) Western blot reveals a specific band that reacts to anti-Siglec-G
Cell number (% max) Cell number (% max)
Fig. S4. Siglec-10Fc reacts with both Siglecg+/+ and Siglecg-/- spleen
cells. Spleen cells from Siglecg +/+ and Siglecg -/- mice were incubated
with biotinylated CD24-Fc or Fc control (2 μg/ml). After washing away the
unbound proteins, the cell-associated proteins were detected by
phycoerythorin-conjungated streptavidin. The FACS stainings have been
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