FIGURE S1 Macrophages respond accordingly to the intensity of TLR stimuli. Peritoneal macrophages were treated with different doses ofLPS (0, 0.5, 2, 8, 32 or 128 ng/ml) for 4.5h, Poly (I:C) (0, 5, 10, 20, 40 or 80 mg/ml) for 5h, or CpG ODN (0, 1, 2, 4, 8 or 16 mg/ml) for 6h. Thelevels of IL-1b (a), iNOS (b), CCL5 (c) or CXCL10 (d) were determined by quantitative PCR. Data are shown as mean 6 S.E.M. of threeindependent experiments.

FIGURE S2 Extracellular Ca21 is required for TLR-triggered response in macrophages. Peritoneal macrophages were treated with LPS (1 ng/ml),Poly (I:C) (10 mg/ml), or CpG ODN (5 mg/ml) for 6h. The RPMI 1640 medium was incubated with or without (-) 1 mM EGTA for 2h to remove Ca21

before adding Ca21 (2 mM) and TLR ligands as indicated. The levels of IL-1b (a), iNOS (b), CCL5 (c) or CXCL10 (d) were determined byquantitative PCR. Data are shown as mean 6 S.E.M. of three independent experiments. *p , 0.05; **p , 0.01; ***p , 0.001.

Cellular & Molecular Immunology

FIGURE S3 Knockdown of STIM1 impairs innate response of macrophages upon TLR stimuli. Peritoneal macrophages were treated with LPS(1 ng/ml), Poly (I:C) (10 mg/ml), or CpG ODN (5 mg/ml) for 6h after STIM1 knockdown. The levels of IL-1b (a), iNOS (b), CCL5 (c) or CXCL10(d) were determined by quantitative PCR. Data are shown as mean 6 S.E.M. of three independent experiments. **p , 0.01; ***p , 0.001.

Cellular & Molecular Immunology

FIGURE S4 Knockdown of STIM1 impairs innate response of macrophages upon TLR stimuli. (a) Peritoneal macrophages were transientlytransfected with control (Ctrl) or STIM1-specific siRNA. After 48h, efficiency of STIM1 knockdown was examined by Western blot. Peritonealmacrophages were treated with LPS (1 ng/ml), Poly (I:C) (10 mg/ml), or CpG ODN (5 mg/ml) for 6h after STIM1 knockdown, The levels of IL-6 (b),TNFa (c), IL-1b (f), iNOS (g), CCL5 (h) or CXCL10 (i) were determined by quantitative PCR and ELISA (supernatant; d, e). Data are shown as mean6 S.E.M. of three independent experiments. **p , 0.01; ***p , 0.001.

Cellular & Molecular Immunology

FIGURE S5 Knockdown of STIM1 decreases the strength of Ca21 influx of macrophages upon TLR stimuli. Peritoneal macrophages pre-labelled byCal-520 were primed with LPS (100 ng/ml), Poly (I:C) (80 mg/ml), or CpG ODN (40 mg/ml) for 3 min; then, 2mM Ca2+ were added and Ca2+ influxwas detected by flow cytometry (a-c). Data are representative of three independent experiments.

Cellular & Molecular Immunology

FIGURE S6 Removal of extracellular Ca21 inhibits TLR-triggered response in Rap1a-silenced macrophages. Peritoneal macrophages weretransfected with control siRNA (Ctrl) or Rap1a siRNA for 48h, and then treated with LPS (1 ng/ml), Poly (I:C) (10 mg/ml), or CpG ODN (5 mg/ml) for 6h. EGTA (2 mM) was incubated with medium for 2h to remove the initial Ca21 before adding TLR ligands. The IL-6 (a, c) and TNFa (b, d)levels were measured by quantitative PCR (a, b) and ELISA (supernatant; c, d). Data are shown as mean 6 S.E.M. of three independentexperiments (a, b) or mean 6 S.D. of triplicate samples (c, d). ***p , 0.001.

Cellular & Molecular Immunology

FIGURE S7 Extracellular Ca21 potentiates TLR-triggered response in human monocyte-derived macrophages. Human MDM were treated withLPS (1 ng/ml), Poly (I:C) (10 mg/ml), or CpG ODN (5 mg/ml). EGTA (2 mM) was incubated with medium for 2h to remove the initial Ca21 beforeadding Ca21 (0, 1, 2 or 4 mM) and TLR ligands. The levels of TNFa (a) IL-1b (b), iNOS (c), CCL5 (d) or CXCL10 (e) were determined by quantitativePCR. Data are shown as mean 6 S.E.M. of three independent experiments. ***p , 0.001.

Cellular & Molecular Immunology

FIGURE S8 Removal of extracellular Ca21 inhibits TLR-triggered response in human monocyte-derived macrophages. Human MDM were treatedwith LPS (1 ng/ml), Poly (I:C) (10 mg/ml), or CpG ODN (5 mg/ml) for 6h. The RPMI 1640 medium was incubated with EGTA (0, 1, 2 and 4 mM) for 2hto remove Ca21 before adding TLR ligands. The levels of TNFa (a) IL-1b (b), iNOS (c), CCL5 (d) or CXCL10 (e) were determined by quantitativePCR. Data are shown as mean 6 S.E.M. of three independent experiments. ***p , 0.001.

Cellular & Molecular Immunology