Mariathasan et al.

Supplementary Figure Legends and Supplementary References

Supplementary Figure 1. Targeted disruption of the murine asc gene. (a) Exons (closed boxes) encompassing the entire asc coding sequence were replaced with a neomycin resistance cassette. (b) Southern blot analysis of offspring from asc+/- matings. Eco RV-digested tail DNA was hybridized with the probe indicated in (a) to yield an 11.3 kb wild-type and a 6.4 kb asc mutant band. (c) Western blot analysis of ASC expression in thioglycollate-elicited peritoneal macrophages from asc+/+, asc+/-, and asc-/- mice (upper panel). The same blot was reprobed for -actin to control for protein loading (lower panel).

We generated two independent lines (1B8 and 1H10) of ASC-deficient mice. Despite high expression of ASC in mouse embryonic heart, liver and brain 1, asc-/- mice were born at the expected Mendelian frequency from asc+/- matings. Histological evaluation of 15-week-old asc-/- mice did not reveal abnormalities in any of the major organs (data not shown) and absolute numbers of B-cells, T-cells, macrophages, and granulocytes in the thymus, spleen, and lymph nodes of asc-/- mice were within normal limits (data not shown). Both lines of asc-/- mice had the same phenotype indicating that ASC is dispensable for normal mouse development.

Supplementary Figure 2. LPS stimulation is not necessary for caspase-1 activation by S. typhimurium. Thioglycollate-elicited peritoneal macrophages from asc+/+ mice were stimulated with or without 500 ng/mL LPS for 16 hours and then infected with wild-type SL1344 S. typhimurium (lanes 3 and 4). Cell lysates were immunoblotted with antibodies against the p10 subunit of caspase-1.

Supplementary Figure 3. Association of ASC and caspase-1 was not detected in macrophage cell lysates. Thioglycollate-elicited peritoneal macrophages from asc+/+ and asc-/- mice were stimulated with 500 ng/mL LPS for 16 hours and then infected with S. typhimurium for the times indicated. Caspase-1 was immunoprecipitated from cell lysates with antibodies to the p10 subunit but Western blotting with antibodies to ASC failed to detect coprecipitating ASC. Control immunoprecipitations were performed with antibodies to mouse EDAR (control IgG), a protein not expressed by macrophages. Whole cell lysates (the two lower left panels) and 30 uL of culture supernantant (from 1 mL cultures; the two lower right panels) also were immunoblotted for ASC and the p10 subunit of caspase-1. Significant accumulation of both caspase-1 and ASC was seen in the supernatants.

Supplementary Figure 4. Both TLR stimulation and extracellular ATP are required for caspase-1 activation and IL-1 release by macrophages. (a-b) Thioglycollate-elicited peritoneal macrophages from asc+/+ and asc-/- mice were stimulated with or without LPS (500 ng/mL) for 16 hours and then pulsed with or without 5 mM ATP for 20 minutes. (a) Cell lysates were immunoblotted with antibodies against the p10 subunit of caspase-1. (b) ELISA measurements of IL-1 secreted into the culture supernantant. (c-d) Thioglycollate-elicited peritoneal macrophages from ipaf+/+ and ipaf-/- mice were stimulated with or without LPS (500 ng/mL) for 16 hours. (c) Cell lysates were immunoblotted with antibodies against the p10 subunit of caspase-1. (b) ELISA measurements of IL-1 secreted into the culture supernantant.

Supplementary Figure 5. Defective secretion of IL-1 by asc-/- macrophages is due to defective maturation of pro-IL-1 rather than impaired release of mature IL-1. Thioglycollate-elicited peritoneal macrophages from asc+/+ and asc-/- mice were stimulated with or without LPS (500 ng/mL) for 3 hours and then pulsed with [35S]-methionine for 45 minutes. Cells were treated or not with ATP for 30 min and then lysed or incubated a further 17.5 hours. Immunoprecipitation and Western blotting was performed on cell lysates using antibodies to IL-1. A small amount of mature IL-1 (indicated by an asterisk) was detected in wild-type cells treated with ATP for 30 minutes but not in asc-/- cells. Consistent with previous studies 2, the majority of mature IL-1 always was detected in the supernatant of wild-type cultures (Fig. 1c).

Supplementary Figure 6. ASC-deficient mice challenged with LPS and ATP produce less IL-1, IL-1, and IL-18 than their wild-type counterparts. asc+/+ and asc-/- mice were injected intraperitoneally with 50 mg/kg of LPS and serum levels of IL-1aIL-18 (b), IL-1c) and TNF (d) were determined after 3.5 hours by ELISA. Mice received an additional 50 L intraperitoneal injection of 100 mM ATP 15 minutes prior to being bled to induce maximal IL-1 release. A control group of asc+/+ mice did not receive LPS. Data represent the mean + standard deviation for 4 mice of each genotype.

When ATP was not injected into asc+/+ mice, IL-1 and IL-18 levels in the serum at 3.5 hours after LPS challenge were below the limits of detection by ELISA. Consistent with a previous study 3, intraperitoneal injection of ATP raised serum IL-1 and IL-18 levels into a detectable range.

Although TNF secretion by cultured asc-/- macrophages was comparable to that of wild type cells (Fig. 2f), TNF production in the whole animal showed a modest diminution (Supplementary Fig. 6d). This reduction in TNF is likely secondary to impaired IL-1 and IL-18 secretion because these cytokines also induce TNF production in various cell types. Indeed, caspase-1-deficient mice also showed decreased serum TNF following LPS challenge 2. Our results demonstrate that ASC, like caspase-1, is an important mediator of LPS-induced endotoxic shock.

Supplementary Figure 7. ASC is dispensable for normal LPS- and TNF-induced IKK and ERK activation. Thioglycollate-elicited peritoneal macrophages from asc+/+ and asc-/- mice were stimulated with either LPS (100 ng/mL) (a) or TNF (20 ng/mL) (b). Cell lysates harvested at the time points indicated were immunoblotted for phospho-IB, total IB-, phospho-ERK, and total ERK.

Supplementary Figure 8. RIP2 is dispensable for caspase-1 activation. (a-c) Thioglycollate-elicited peritoneal macrophages from rip2+/+ and rip2-/- mice were stimulated with 500 ng/mL LPS for 16 hours and then infected with S. typhimurium for the times indicated. Cell lysates were immunoblotted with antibodies against the p10 and p20 subunits of caspase-1 (a). Culture supernatants were harvested for immunoprecipitation and Western blotting with antibodies that recognize both pro-IL-1 and mature IL-1(b). Cell death in infected cultures was measured by LDH release andis shown as a percentage of LDH release by detergent (c). (d-e) Thioglycollate-elicited peritoneal macrophages from rip2+/+ and rip2-/- mice were stimulated for 16 hours with HKLM (108 bacteria/mL), LTA (1 g/mL), PGN (10 g/mL), LPS (100 ng/mL), R848 (100 ng/mL) or media alone (unstim). Cells were then pulsed transiently with ATP for 20 minutes (see methods and materials). IL-1(d and IL-18 (e) released into the media in the 3 hours after ATP treatment were measured by ELISA.

Supplementary Figure 9. Generation of Ipaf-deficient mice. (a) Genomic sequence encoding amino acids 8-374 of Ipaf was replaced with a PGK-neo cassette. (b) Southern blot analysis of offspring from ipaf+/- matings. Nhe I-digested tail DNA was hybridized with the probe indicated in (a) to yield an 11.6 kb wild-type and a 6.4 kb ipaf mutant band. (c) Immunoprecipitation and Western blot analysis of Ipaf in thioglycollate-elicited peritoneal macrophages from ipaf+/+ and ipaf-/- mice. Immunoprecipitations were performed with rabbit polyclonal antibodies and Western blots used a hamster monoclonal antibody against amino acids 610-826 of mouse Ipaf.

These antibodies immunoprecipitated and Western blotted full-length Ipaf (116 kDa) from ipaf+/+ but not ipaf-/- macrophages.

Supplementary Figure 10. Expression of ASC and Ipaf is not affected in Ipaf-null macrophages and ASC-null macrophages, respectively. Lysates from wild type (WT), asc-/- and ipaf-/- macrophages were immonoblotted with antibodies against caspase-1, Ipaf, ASC and actin before and after infection with S. typhimurium.

1.Masumoto, J., Taniguchi, S., Nakayama, K., Ayukawa, K. & Sagara, J. Murine Ortholog of ASC, a CARD-Containing Protein, Self-Associates and Exhibits Restricted Distribution in Developing Mouse Embryos. Exp Cell Res262, 128-133 (2001).

2.Li, P. et al. Mice deficient in IL-1 beta-converting enzyme are defective in production of mature IL-1 beta and resistant to endotoxic shock. Cell80, 401-11. (1995).

3.Griffiths, R. J., Stam, E. J., Downs, J. T. & Otterness, I. G. ATP induces the release of IL-1 from LPS-primed cells in vivo. J Immunol154, 2821-8 (1995).

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