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Figure S1. IL-33 treatment of mast cells results in insensitivity to LPS and PGN. (A and B) BMMCs were treated for 24 h with media alone or 1 ng/ml IL-33 then stimulated with 100 ng/ml LPS for 24 h. Supernatant levels of (A) TNFα or (B) CCL2 were assessed. (C-E) BMMCs were treated for 24 h with media alone, 100 ng/ml LPS or 1 ng/ml IL-33, then stimulated with 100 ng/ml LPS or 100ng/ml PMA and 1 μM ionomycin for a further 4 h. (C) IL-6, (D) TNFα or (E) CCL2 mRNA levels are presented relative to β-actin and to the cells stimulated with LPS after pre-treatment in media alone. (F) BMMCs were treated for 24 h with media alone or 1 ng/ml IL-33 then stimulated with 100 ng/ml LPS, 10 μg/ml PGN, 1 μg/ml poly(dA:dT), 5 μg/ml R837, or 5 μg/ml CL075. CCL2 levels in cell supernatants shown. Statistical significance compared to the 1st treatment with media alone. Data shown from (A) 7, (B) 8, (C) 5, (D to F) 6 experiments.

Figure S2. C-kit is not required for the IL-33 induced LPS tolerance. (A) c-kit expression on WT and Kitw-sh/w-sh BMMCs. (B) WT (C) Kitw-sh/w-sh BMMCs were treated for 24 h with media alone, 100 ng/ml LPS or 1 ng/ml IL-33 then stimulated with 100 ng/ml LPS or media alone for a further 24 h. Data shown are from n=3 experiments.

Figure S3. Gating strategies used to identify c-kit high mast cells in peritoneal lavage cells.

Figure S4. TNFα does not cause LPS tolerance in BMMCs. BMMCs were treated for 24 h with media alone (black bars), 100 ng/ml LPS (dark grey bars) or 10 ng/ml TNFα (light grey bars) then stimulated with 100 ng/ml LPS or media alone for a further 24 h. Data shown are from n=3 experiments IL-6 levels in cell supernatants were assessed.

Figure S5. Expression of A20 is increased by short IL-33 treatments and Tollip is unaffected by IL-33. (A and B) Immunoblot analysis of whole cell lysates from BMMCs treated with 100 ng/ml LPS, 1 ng/ml IL-33 or media alone for the indicated times with antibodies against A20 or β-actin. (A) Membrane shown is representative of n=4 experiments. (B) Quantification of A20 protein levels relative to β-actin and the media treated cells for 2 h from n=4 experiments. (C) Immunoblot analysis of whole cell lysates from BMMCs treated with 100 ng/ml LPS, 1 ng/ml IL-33 or media alone for the indicated times with antibodies against TOLLIP or β-actin. Data shown is representative of 3 experiments.

Figure S6. IL-33 treatment of mast cells results in IRAK1 degradation and insensitivity to LPS. BMMCs were treated for 24 h with media alone or with 100 pg/ml IL-33 for the indicted time (for example, for the 8 h time point cells were with media alone for 16 h then for a further 8 h with IL-33). (A) Immunoblot analysis of whole cell lysates with IRAK1 antibody. (B) Cells were then stimulated with 100 ng/ml LPS for 24 h and supernatant levels of IL-6 assessed. Data shown are (A) representative of 3 experiments, (B) from n=3 experiments.

Figure S7. Irak1-/- and Irak2-/- BMMCs express similar levels of mast cell markers and TLR4 signalling proteins and respond comparably to stimulation via FcεR1 and PMA/I as WT BMMCs. (A) Flow cytometry of WT, Irak1-/- and Irak2-/- BMMC stained for (A) c-kit, FcεR1 and ST2 or (B) TLR4 (open histogram) or isotype control antibody (filled histogram). (C-E) Immunoblot analysis of whole cell lysates from WT, Irak1-/- and Irak2-/- BMMCs with antibodies against β-actin and (C) Tirap, (D) IRAK1 or MyD88 and (E) IRAK2. (F and G) IL-6 levels were measured in cell supernatants from WT, Irak1-/- or Irak2-/- BMMCs stimulated for 24 h with (F) 100 ng/ml LPS or 10 ng/ml DNP, or (G) 100ng/ml PMA and 1 μM ionomycin.

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