<p>Supp. Figure S1 Caspase-12 targeted deletion. a. Strategy for caspase-12 gene targeted deletion. Murine caspase-12 consists of 10 exons (black boxes) and 9 introns </p><p>(lower case letters). The targeting construct contains a 4 Kb 5’ arm, a lacZ-neo cassette and a 1.5 Kb 3’arm. The homologous recombination replaces a segment of caspase-12 exon 2 by the LacZ-neo cassette and shifts the downstream amino acid sequence out of the reading frame. b. Southern analysis. Genomic DNA isolated from the indicated ES lines was digested with XbaI (determined to cut outside of the construct arms). The DNA was analyzed by Southern hybridization, probing with a radiolabeled DNA fragment that hybridizes outside of and adjacent to the construct arm. The parent ES lines (negative controls) showed bands representing the endogenous (wild-type) allele. In contrast, the </p><p>ES#2495 line showed an additional band representing the targeted allele from the expected homologous recombination event. c. Western analysis of protein extracts obtained from intestinal epithelial cells and probed with anti-caspase-12 antibodies showing caspase-12 expression in caspase-12+/+ but not in caspase-12-/- extracts. In vitro transcribed and translated rat caspase-12 was used as a positive control. d, f Expression of caspase-11 and caspase-1, respectively, in mouse peripheral blood was examined in response to LPS treatment using Western analysis. e. quantitative real-time PCR showing intact caspase-11 expression and induction by LPS in peripheral blood of both caspase-12+/+ (n=3) and caspase-12-/- mice (n=3).</p><p>Supp. Figure S2 TNF and IL-6 levels are not modulated by caspase-12 in response to </p><p>TLR/NOD2 ligands see also Fig. 2).</p><p>Supp. Figure S3 Unlike the human Csp-12L variant, rodent caspase-12 does not inhibit NF-B signaling. a. HEK 293T cells were co-transfected with pRSV-gal and pB-luc reporter plasmids along with either pcDNA3.1 vector control, pcDNA3.1-human Csp-</p><p>12L or pcDNA3.1-rat caspase-12, and relative B–luc activity was measured in response to TNF. Results are representative of three independent experiments. b. Mouse embryonic fibroblasts from Csp-12+/+ or Csp-12-/- mice were transfected with pRSV-gal and pB-luc reporter plasmids and relative B–luc activity was measured in response to </p><p>TLR ligands.</p><p>Supp. Figure S4 The role of caspase-12 in bacterial clearance is not restricted to the hematopoeitic system. Reconstituted bone marrow chimeras were subjected to CASP- induced sepsis, and clearance of the polymicrobial infection was assessed as in Fig. 1c, showing that bone marrow from caspase-12+/+ mice ablated the ability of caspase-12-/- mice to clear bacteria, while that of caspase-12-/- mice only partially improved the clearance ability of caspase-12+/+ mice. Csp-12+/+, Csp-12-/-, controls: n=5; Csp-12+/+ recipient of -/- bone marrow: n=9; Csp-12-/- recipient of +/+ bone marrow: n=10.</p><p>Supp. Figure S5 Caspase-12 functions independently of NOD2 downstream of TLR </p><p> signaling. Csp-12+/+ or Csp-12-/- splenocytes were treated with the TLR2 ligand </p><p>PAMcsk4 only or in combination with the NOD2 ligand MDP. Levels of IFN (a) </p><p> and IL-6 (b) were measured from the culture media using bead-based immunoassays </p><p> as in Fig. 2.</p><p>Supp. Figure S6 Caspase-12 does not mediate ER-stress apoptosis. Mouse embryonic fibroblasts from caspase-12+/+ and caspase-12-/- mice were treated for 24 hrs with the indicated concentrations of ER stressors, and cell death was measured by quantification of propidium iodide (PI) uptake using flow cytometry. Treatments were done in triplicates. Results are representative of four independent experiments.</p>