Supplemental Figure 1.

Transcription

1 antifas CD40L min piκbα IκBα p38 Supplemental Figure 1. CD40L and antifas stimulation induces NFkB activation. BMDM were stimulated with CD40L (1ug/ml) or antifas (10ug/ml). Cell extracts were collected at the indicated times and immunoblotted with antibodies that detect total and phosphorylated IκBa or p38. Results are representative of three separate experiments.

2 A ATP min. B ng/ml Supplemental Figure 2. αdependent activation of caspase1. A, BMDM were stimulated for 6 hrs with (100 ng/ml) and then stimulated with the indicated doses of ATP. B, BMDM were stimulated for 6 hrs with the indicated doses of and then stimulated with ATP (5 mm) for the last 30 minutes Extracts were prepared from cell and culture supernatants and immunoblotted with caspase1 antibody. Arrows denote procaspase1 () and its processed subunit. Results are representative of three separate experiments.

3 IL1α IL1α IL1α IL1α IL1α IL1α WT Nlrp3KO AscKO Supplemental Figure 3. ATPinduced caspase1 activation in macrophages stimulated with α or IL1α is dependent on Nlrp3 and ASC. BMDM from WT, Nlrp3KO and AscKO mice were stimulated with α or IL1α for 6 hrs and then stimulated, or not, with ATP for 30 minutes. Extracts were prepared from cell and culture supernatants and immunoblotted with caspase1 antibody. Arrows denote procaspase1 () and its processed subunit. Results are representative of three separate experiments.

4 IL1β (pg/ml) WT RI/IIKO Unst +ATP Supplemental Figure 4. induced IL1β production is independent of RI/II. BMDC from WT and RI RII DKO mice were stimulated with for 6hrs and then stimulated, or not, with ATP (5 mm) for 30 minutes. Cellfree supernatants were analyzed by ELISA for production of IL1β 4 hrs after stimulation. Values represent mean ± s.d. of triplicate cultures. Results are representative of three separate experiments.

5 WT P2X7RKO Supplemental Figure 5. ATPinduced caspase1 activation in macrophages stimulated with α is P2X7R dependent. BMDM from WT and P2X7RKO mice were stimulated with α or for 6 hrs and then stimulated, or not, with ATP for 30 minutes. Extracts were prepared from cell and culture supernatants and immunoblotted with caspase1 antibody. Arrows denote procaspase1 () and its processed subunit. Results are representative of three separate experiments.

6 min p38 pp38 ERK1/2 perk1/2 IκBα WT Nlrp3KO Supplemental figure 6. Nlrp3 is not involved in α induced NFκB and MAPK activation. BMDM from WT and Nlrp3 KO mice were stimulated with a (50 ng/ml) and cell extracts were immunoblotted with antibodies that detect total and phosphorylated (activated) forms of the indicated proteins. Extracts were immunoblotted with caspase1 antibody. Results are representative of three separate experiments.

7 Salm Salm CHX Supplemental figure 7. Salmonellainduced caspase1 activation does not require gene transcription. BMDM were pretreated with CHX for 1 hr and then infected for 1 hr with S. Typhimurium at a macrophage/bacterial ratio of 1/10. Extracts were prepared from cell and culture supernatants and immunoblotted with caspase1 antibody. Arrows denote procaspase1 () and its processed subunit. Results are representative of three separate experiments.

8 A WT TLR4KO B ATP ATP ATP ATP CHX BAY Supplemental Figure 8. promotes caspase1 activation via TLR4 and gene transcription. A, BMDC from wildtype (WT) and TLR4KO were stimulated with or a for 6 hrs and then stimulated, or not, with ATP for 30 minutes. B, BMDM were pretreated with 50 μm of cyclohexemide (CHX) or the NFkB inhibitor BAY (20 μm) for 1 hour, and then stimulated with (100ng/ml) for 2 hrs. When indicated ATP was added for the last 30 minutes. A and B, Extracts were immunoblotted with caspase1 antibody. Arrows denote procaspase1 () and its processed subunit. Results are representative of five (A) and three (B) separate experiments.

9 min ERK1/2 perk1/2 IκBα pretreatment Supplemental Figure 9. Pretreatment with α renders macrophages tolerant to successive α stimulation. BMDM were pretreated for 24 hrs with α (100 ng/ml) and then stimulated with α (100 ng/ml). Cell extracts were collected at the indicated times and immunoblotted with antibodies that detect total and phosphorylated (activated) forms of ERK1/2. or IκBα. Notice impaired ERK phosphorylation and IκBα degradation in pretreated cells. Results are representative of at least three separate experiments.

10 IL6 (ng/ml) unstimulated tolerized tolerized ctr BLP CpG (ng/ml) 4,5 4 3,5 3 2,5 2 1,5 1 0,5 0 unstimulated tolerized tolerized ctr BLP CpG Supplemental Figure 10. Pretreatment with α renders macrophages tolerant to successive stimulation with TLR ligands. BMDM were pretreated for 24 hrs with α (100 ng/ml) or (100 ng/ml) and then stimulated with (100 ng/ml), BLP (100 ng/ml), or CpG (10 μg/ml) for 6 hrs. Cellfree supernatants were analyzed by ELISA for production of and IL6. Values represent mean ± s.d. of triplicate cultures. Results are representative of three separate experiments.

11 A IL1β (pg/ml) ATP 10 0 Unst. 4hrs 24 hrs B IL1β (pg/ml) ATP n.s Unst. 4hrs 24 hrs Supplemental figure 11. Prolonged stimulation with α, but not, renders human monocytes sensitive to ATP. Human monocytes were stimulated for the indicated time point with or. Where indicated ATP 5 mm was added for the last 30 minutes. Cellfree supernatants were analyzed by ELISA for production of IL1β. Results are representative of four experiments performed on monocytes from different donors.