Supporting Information
Supplementary Figure 1 (a) Jurkat T cells were treated with 3 µM of dexamethasone
(Dexa) for 24 h, and apoptosis induction was confirmed by analyzing the
immunostaining of Annexin-V and propidium iodide (PI) by FACS analysis, resulting in
>70% of apoptotic cells. (b) As in (a), but mouse thymocytes were used. (c) As in (a),
but apoptosis was induced in mouse thymocytes by exposure to -irradiation (1,500 rad),
which was followed by a 4 h culture in RPMI-1640/0.4% BSA. All of these results
showed that there were no PI-single positive necrotic cells with above experimental
conditions.
Supplementary Figure 2 Different area of Figure 2c (10 × magnification) is presented
to demonstrate apoptotic cell binding to DCEK_WT or _SIGN-R1, and insets were
enlarged below. Arrows indicate apoptotic cells bound to DCEKs.
Supplementary Figure 3 (a) Mice were injected i.v. with 0.5 or 2 μm size of TRITCconjugated latex beads (red) into mice. 1 h later, tissues of spleen, thymus and liver
were harvested and immunostained with SIGN-R1 (green). (b) 1  107 apoptotic
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thymocytes (green) were intravenously injected into control and SIGN-R1 KO mice for
30 min or 1h, and spleen sections were immunostained for SIGN-R1 (blue). Apoptotic
cells were counted in 5 areas of each spleen section from 3 independent experiments,
and average numbers were calculated (right).
Supplementary Figure 4 (a) DCEKs and CFSE-apoptotic thymocytes were
coincubated at a 1:4 ratio, with or without 10% normal mouse serum for 30 min at
37 °C. After extensive washes, cells were analyzed by a forward scatter detector (FSC)
and a side scatter detector (SSC) by FACS analysis, and a single-cell region was gated
out (left). Cells in the single-cell region were further analyzed by a FSC and a
fluorescent detector for CFSE in FACS analysis in order to differentiate between
DCEKs only (Region 1, R1) and apoptotic cell-bound DCEKs (Region 2, R2) (right).
(b) Original image of Figure 5b (10X magnification) shows C3 deposition on apoptotic
cells in the splenic MZ, and insets were enlarged in Figure 5b. (c) Three different areas
of Figure 5b are presented to confirm C3 deposition on apoptotic cells in the splenic MZ
(right or far right). (d) Different areas of Figure 5b are presented to confirm C3
deposition on apoptotic cells in the splenic MZ with another anti-C3 antibody (ICN
Pharmaceuticals). The enlarged area is marked on the original 20X power image (inset,
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far right). (e) The sera of control or cobra venom factor-injected mice, which were used
in Figure 5d, were immunoblotted with anti-C3 antibody to confirm the depletion of C3.
(f) Original 10X power image of Figure 5d is presented to demonstrate the
disappearance of C3 deposition on apoptotic cells, and the inset was enlarged in Figure
5b. (g) Three different areas of Figure 5d are presented to confirm the inhibition of C3
deposition on apoptotic cells in the splenic MZ. The enlarged areas are marked on the
original 10X power image (right). (h) Original 10X power image of Figure 5e is
presented to show the disappearance of C3 deposition on apoptotic cells, and the in-box
was enlarged in Figure 5e. (i) Three different areas of Figure 5e are presented to confirm
the inhibition of C3 deposition on apoptotic cells in the splenic MZ of SIGN-R1 KO
mice. Splenic MZs are marked with white dotted lines.
Supplementary Figure 5 (a) Seven different areas of Figure 6a. The 1st row are
presented to confirm C3 deposition on apoptotic cells in the liver of control mice. (b)
Liver section of Figure 6a, the 1st row is immunostained with nonreactive rabbit IgG as
a C3-staining specificity. (c) Two different areas of Figure 6a, the 2nd row are presented
to confirm the inhibition of C3 deposition on apoptotic cells in the liver of C3-depleted
mice. The enlarged areas are marked on the original 10X power image (right). (d)
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Different areas of Figure 6a, the 3rd row is presented to confirm the inhibition of C3
deposition on apoptotic cells in the liver of SIGN-R1 KO mice. The enlarged areas are
marked in the original 10X power image (right). (e) Representative 40X power image
for Figure 6a is presented. The number of C3-deposited apoptotic cells (red)(white
dotted circles) of total apoptotic cells (green) was counted from the livers of control,
C3-depleted, and SIGN-R1 KO mice.
Supplementary Figure 6 (a) 1  107 apoptotic thymocytes (green) were intravenously
injected into control and SIGN-R1 KO mice for 30 min or 1h, and spleen sections were
immunostained for SIGN-R1 (blue). Apoptotic cells were counted in 5 areas of each
spleen section from 3 independent experiments, and average numbers were calculated
(right). (b) The preferential entrapment and phagocytosis of apoptotic cells by F4/80+
Kupffer cells in liver. CFSE-labeled apoptotic thymocytes (1  108; green) were injected
into control mice for 30 min, and the liver section was immunostained for F4/80 (red).
(c) Wild-type or SIGN-R1 KO mice were intravenously injected with PBS or 5  107
apoptotic cells for 1 h (black or empty bars, respectively), and the endogenous levels of
IL-10 were examined from tissue lysates of spleens or livers by ELISA. The bars are the
mean values for the 7 mice per group. (d) MRL/MpJ mice were intravenously injected
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with 100 g of nonreactive hamster IgG or 22D1 anti-SIGN-R1 mAb for 24 h
(MRL/MpJ_hamster IgG or MRL/MpJ_SIGN-R1 TKO, respectively), which was
followed by PBS or 4 intravenous injections of apoptotic thymocytes (107 cells/mouse)
with 1-week intervals. Sera from MRL/MpJ_hamster IgG or MRL/MpJ_SIGN-R1 TKO
mice were collected at the indicated time points and assessed for the presence of antissDNA Ab (IgG). The data shown are mean ± SEM and representative plots of 2-3
experiment, in which the error bars represent SEM (n = 5 mice per group).
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