(A) Real time PCR analysis for mRNA of Nlrp12 in macrophages, dendritic cells (DC), PMN and T cells collected from wild-type mouse. (B) Real time PCR analysis for mRNA of Nlrp12 in liver, spleen, small intestine (SI), cecum, colon and MLN. (C, H) Wild-type and Nlrp12^−/− mice were fed a 3% DSS-solution in drinking water for 6 days followed by regular drinking water until the end of the study. (C) Body weight, (D) clinical score (stool consistency plus rectal bleeding score) were scored daily. Data represent means ± SEM; n=20/group; _*, p<0.05; _{* *}, p<0.01. (E, F) Mice were sacrificed on day 5 and 9 to measure colon length. Histopathological changes in colon tissue collected at day 5, 9, and 20 were examined by H&E staining. (G) Semi-quantitative scoring of histopathology. (H) Representative images of H&E staining of colon section at day 0, 5, 9 and 20 after DSS administration. Scale bar; 50 μM. Data represent means ± SEM. _*, p<0.05; _{* *}, p<0.01. See also .

(A) WT (n=16), Nlrp12^−/− (n=14), were injected with AOM on day 0 (d0), and were then given a 3% DSS solution during three 5-day cycles as described in Methods. (B) Body weight changes were monitored throughout the study. (C) 12 weeks after AOM injection, mice were sacrificed to determine tumor development in the colon. Number of tumors in the whole colon was counted (D), and diameter of the tumors (E) was measured at the end of the study. At the same time colons were processed for histopathological analysis. (F–G) H&E stained sections were scored for inflammation, ulceration, hyperplasia and inflamed area. (H) Overall grading of dysplasia in each genotype. (I) Representative high magnification images showing dysplasia in colon tissue. Scale bar; 100 μM. Data represent means ± SEM; ^*, p<0.05, ^** p<0.01. See also .

Wild-type and Nlrp12^−/− mice were injected with AOM and 5 days later mice were fed with 3% DSS in drinking water for 5 days. Distal colons were collected at day 15 after AOM injection and examined after H&E staining. (A) Representative images of H&E stained sections of distal colon. Scale bar; 200μM. (B) Total histological scores of whole colon at the same time. Data represent means ± SEM; n=5/group; ^*, p<0.05. (C) Colon tissue was immunostained for the macrophage marker F4/80, PMN marker Gr-1 and T cell marker CD3. Scale bar; 100μM. (D) F4/80 staining in non-inflamed part of the colon at day 10 after DSS. Scale bar; 20μM. (E–G) In a separate experiment, mice were fed with 3% DSS for 5 days followed by normal drinking water until day 20. Colon, MLN and spleen were collected at day 5, 9, and 20 after DSS. Colonic lamina propria cells, MLN cells, and splenocytes were analyzed flow cytometrically after staining for myeloid cell markers CD11b, F4/80, CD11c and Gr-1. Total number of CD11b⁺, F4/80⁺, CD11c⁺, and Gr-1⁺ cells at day 5 (E), day 9 (F), and day 20 (G). Data represent means ± SEM; n=5/group; ^*, p<0.05; ^**, p<0.01. See also .

WT and Nlrp12^−/− mice were fed with 3% DSS for 5 days followed by normal drinking water until day 20. MLN and spleen were collected at day 20 after DSS administration and cells were stimulated with either LPS (1 μg/ml) or PMA (25ng/ml) plus ionomycin (500ng/ml) for 5h. After stimulation cells were stained for myeloid cell marker CD11b, and intracellular cytokines IL-6 and TNFα. (A) Frequency of CD11b⁺TNFα⁺ and CD11b⁺IL-6⁺ cells. (B) Mean fluorescent intensity (MFI) of IL-6⁺CD11b⁺ and TNFα ⁺ CD11b⁺ cells in LPS stimulated MLN and spleen. Data represent means ± SEM; n=5/group; ^*, p<0.05, ^** p<0.01. In a separate experiment, wild-type and Nlrp12^−/− mice were injected with AOM and 5 days later mice were fed with 3% DSS in drinking water for 5 days. (C) Whole colons were collected at day 14 and day 18 after AOM injection and homogenates were used to determine cytokines by multiplex ELISA. Data represent means ± SEM; n=7–8/group; ^*, p<0.05, ^** p<0.01.

Wild-type and Nlrp12^−/− mice were injected with AOM and 5 days later mice were fed with 3% DSS in drinking water for 5 days. (A) Distal colons collected at day 15 after AOM injection were used to isolate RNA for expression analysis of IL-6, TNF-α, IFN-γ, MIP2, COX2 and NOS2 by real time PCR. Data represent means ± SEM; n=5–7/group; ^*, p<0.05, ^** p<0.01. (B) Real time PCR analysis for COX2 and MIP2 in distal colon tumors collected at 12 weeks after AOM/DSS treatment. Data represent means ± SEM; n=5–7/group;^** p<0.01. (C) Whole colon tissue homogenates collected at 15 days after AOM injection (10 days after DSS) were examined for activation of NF-κB, p38, ERK and STAT3 by Western blot analysis. Each lane corresponds individual mouse. (D) Densitometric analysis of pIκBα, p-ERK and p-STAT3 relative to their total protein. Data represent means ± SD; n=5/group; ^*, p<0.05. In a separate experiment, mice were injected i.p. with BrdU either at day 15 or 12 weeks after AOM injection. Colon sections were stained for BrdU-positive cells and macrophages. (E) Representative images of BrdU staining at 15 days and 12 weeks after tumor induction. Scale bar; 50μM (F) Number of BrDU-positive cells at day 10 after DSS treatment (day 15 after AOM). Data represent means ± SEM; n=4 mice/group; 3 different section per mouse; 50 field per section; ^*, p<0.05. (G) Serial sections of Nlrp12^−/− mouse colon at day 10 (day 15 after AOM) were stained for macrophage marker F4/80 (lower panel) and BrdU-positive cells (upper panel). Scale bar; 20μM. See also .

Bone marrow chimera mice for Nlrp12 were generated as described in Methods. 8 weeks after, mice (8–10/group) were induced tumorigenesis using AOM/DSS regimen as described in Methods. (A) Body weight was monitored every alternate day. (B–C) Tumor burden in whole colon and length of colon were measured at 12 weeks after tumor induction. Data represent means ± SEM; ^*, p<0.05. (D) In a separate study, colon tissue collected at 15 days after AOM injection were processed for Western blot analysis for IκB, p-IκB, ERK and p-ERK. (E) Densitometric analysis of p-IκBα and p-ERK relative to their total protein. Data represent means ± SD; n=3/group; ^*, p<0.05. ^**, p<0.01.

Bone marrow derived macrophages were cultured as described in Methods and were stimulated with (A) LPS (1 μg/mL), (C) Pam3-CSK4 (5 μg/mL) (E) poly(I:C) (1 μg/mL). Cell lysates collected at indicated time points were analyzed for p-IκB, I-κB, p-P38, pERK, ERK, p-JNK and JNK by Western blotting. Densitometric analysis of band intensity of p-IκBα and p-ERK relative to IκBα and ERK of LPS (B), Pam3CSK4 (D) and Poly(I:C) (F) treated cells. (G) LPS-stimulated wild-type and Nlrp12-deficient macrophages were collected at different time points and mRNA was isolated for real-time quantitative PCR analysis of IL-6, KC, TNF-α, MIP2, COX2 and NOS2 synthesis. Data represent means ± SD of triplicate wells; ^*, p<0.05; ^**, p<0.01. See also .

Bone marrow derived macrophages from wild-type and Nlrp12^−/− mice were stimulated with LPS (1 μg/mL). Total cell lysate (A) and cytoplsmic fraction (B) were analyzed for phosphorylation of P100 and P105 by Western blotting. (C) Western blot analysis of NF-κB complex component RelA and RelB in nuclear fraction. (D) 5 μg nuclear protein was assayed for p65 DNA binding activity using commercial ELISA kit as described in Methods. Absorbance at 450 nm represents corresponding DNA binding activity of P65 (RelA) in the nuclear extract. Data represent means ± SD of triplicate wells in ELISA assay of a single sample; ^**, p<0.01.