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Supplementary Fig. S1. Identification of macrophages and neutrophils. (A) The
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macrophages were identified by immunofluorescence staining using antibodies
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against F4/80 (a marker for macrophages). (B) Purity and viability of macrophages.
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The cells were analyzed by flow cytometry after double staining with
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FITC-conjugated annexin Ⅴ (FITC-AnxV) and phycoerythrin-conjugated antibodies
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against F4/80 (PE-F4/80 Ab). The viable macrophages were classified under an F4/80
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＋
/AnxV－ population (left). The cells labeled with PE-conjugated rat immunoglobulin
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G2a (IgG2a) were used as isotype control (right). (C) Identification of neutrophils.
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Mouse neutrophils were isolated from bone marrows, and stained with Wright’s
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Giemsa. The living cells before apoptosis induction were characterized by multilobed
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nuclei (left), whereas the apoptotic cells at 24 h after culture in serum-free medium
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were recognized by apoptotic bodies (right). (D) Purity and apoptosis rate of
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neutrophils. Before and after apoptosis induction, the cells were co-stained with
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FITC-AnxV and propidium iodide (PI), and then analyzed by flow cytometry.
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Supplementary Fig. S2. Effect of poly(I:C) on macrophage phagocytosis. (A)
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Representatives of flow cytometry plots. At 12 h after treatment with 10 μg/ml
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poly(I:C) (right panel) or without poly(I:C) treatment (left panel), macrophages were
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co-cultured with fluorescein isothiocyanate (FITC)-labeled apoptotic cells (FITC-AC)
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for 30 min, and followed by staining with phycoerythrin (PE)-conjugated F4/80
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antibodies (PE-F4/80 Ab). The macrophages that engulfed apoptotic cells were
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quantitatively analyzed by flow cytometry and defined as cells positive for both PE
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and FITC. (B) Time course of phagocytosis. Without poly(I:C) treatment,
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macrophages were co-cultured with apoptotic cells for the indicated duration. The
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number of macrophages that had engulfed apoptotic cells was determined by flow
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cytometry. (C) Flow cytometry analysis. At 20 min after co-culture with GFP-bacteria,
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macrophages were stained with PE-F4/80 Ab and analyzed by flow cytometry.
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Macrophages that engulfed bacteria were defined as cells double positive for F4/80
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and GFP. (D) Time course of bacteria uptake. Macrophages were co-cultured with
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GFP-bacteria for the indicated duration, and analyzed by flow cytometry. The flow
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cytometry plots are representative of one of at least three experiments. Data represent
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the mean ± SEM of three experiments. * P < 0.05; ** P < 0.01.
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Supplementary Fig. S3. Effect of poly(I:C) on the expression of key
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phagocytosis-regulated genes in macrophages. (A) Macrophages of WT (left panel)
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and TLR3-/- (right panel) mice were treated with 10 μg/ml poly(I:C) for 12 h. The
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mRNA levels of the indicated phagocytosis-regulated genes were determined by
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qRT-PCR. (B) Time-dependent effect of poly(I:C) on gene expression. Macrophages
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were treated with poly(I:C) for the indicated time, and the mRNA levels of
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phagocytosis-regulated genes were analyzed by qRT-PCR. (C) Effect of poly(I:C) on
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protein levels of genes. Macrophages were treated with 10 μg/ml poly(I:C) for 12 h.
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Proteins of the phagocytosis-regulated genes were determined by Western blot using
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specific antibodies. Data represent the mean ± SEM of three experiments. * P < 0.05;
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** P < 0.01.
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