Figure S3.
Identification of NLRP6 and generation of NLRP6-deficient mice. (A) Western blot of input samples used in pulldown of Fig. 4 A. β-Actin was used as a loading control. (B) Peptides of NLRP6 were identified by mass spectrometry. (C) Western blot for ASC KO validation with anti-ASC antibody. β-Actin was used as a loading control. (D) Left: Immunofluorescence staining of SIs from sgCtrl and sgAsc mice. Representative Paneth cells and goblet cells are shown. Green: UEA-1, blue: DAPI, and red: EpCAM. Scale bar: 20 μm. Right: Diameter of goblet cell numbers (n = 10 mice and 20 villus-crypts were analyzed per mouse) and DCVs per Paneth cells were quantified (n = 6 mice and 20 villus-crypts were analyzed per mouse). (E) NLRP6 expression levels of small intestinal villi from littermate WT and Cnep1r1ΔIEC mice were detected by western blot with anti-NLRP6 antibody. β-Actin was used as a loading control. (F) Relative Nlrp6 expression levels of intestinal crypts from WT and Cnep1r1ΔIEC mice were detected by qRT-PCR. Fold changes were normalized to endogenous 18S (n = 6 mice for each group). (G) Construction diagram showing generation of Nlrp6−/− mice with CRISPR-Cas9. (H) DNA sequencing for Nlrp6 KO validation. (I) Western blot for NLRP6 KO validation with anti-NLRP6 antibody. β-Actin was used as a loading control for western blot. (J) Left: HE staining of duodenum, jejunum, and ileum from littermate WT and Nlrp6−/− mice. Scale bar: 30 μm. Middle: Proportion of normal and abnormal Paneth cells were quantified based on whether Paneth cells displayed a typical staining pattern with distinguishable granules (normal) or disordered, depleted, and/or diffuse staining (abnormal). Right: Numbers of Paneth cells were quantified. n = 6 mice, with 20 selecting maximal crypt sections that displayed all Paneth cells within each crypt were analyzed per mouse. (K) Quantification of Lyz+ puncta per crypt of WT and Nlrp6−/− mice (n = 6 mice for each group). (L) AB-PAS staining of intestines from WT and Nlrp6−/− mice. Scale bar: 35 μm. (M) Left: 3D immunofluorescence staining of ileum crypts from littermate WT and Nlrp6−/− mice. Green: UEA-1, red: EpCAM, and yellow: DCVs’ surface fitted by Imaris software. Scale bar: 35 μm. Right: Volume of DCVs was quantified (n = 6 mice for each group, and 20 crypts were analyzed per mouse). (N) Immunofluorescence staining of organoids from littermate WT and Nlrp6−/− mice. Green: UEA-1, red: EpCAM, and blue: DAPI. Scale bar: 20 μm. (O) Intestinal lumen (left) and tissue-associated (right) bacterial load analysis, quantified by qPCR of 16S rRNA gene copy number in distal ileums (n = 6 mice for each group). (P) qPCR detection of ileal luminal commensal bacteria classified by phylum (n = 6 mice for each group). (Q) Lysozyme in supernatant of stimulated crypts from littermate WT and Nlrp6−/− mice after treatment of DMSO or CCh was measured by ELISA (n = 6 mice for each group). (R) Bacterial load analysis of S. Typhimurium in secreted supernatants. SI crypts of littermate WT and Nlrp6−/− mice were isolated and stimulated by DMSO or CCh. The crypt secreted supernatants were incubated with S. Typhimurium for 30 min, and CFUs were measured (n = 6 mice for each group). Data in D–F, J, K, M, and O–R are shown as means ± SEM; significance was determined by unpaired two-tailed Student’s t test (*P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant). Data in A–F and H–R are representative of at least three independent experiments. Source data are available for this figure: SourceData FS3.

Identification of NLRP6 and generation of NLRP6-deficient mice. (A) Western blot of input samples used in pulldown of Fig. 4 A. β-Actin was used as a loading control. (B) Peptides of NLRP6 were identified by mass spectrometry. (C) Western blot for ASC KO validation with anti-ASC antibody. β-Actin was used as a loading control. (D) Left: Immunofluorescence staining of SIs from sgCtrl and sgAsc mice. Representative Paneth cells and goblet cells are shown. Green: UEA-1, blue: DAPI, and red: EpCAM. Scale bar: 20 μm. Right: Diameter of goblet cell numbers (n = 10 mice and 20 villus-crypts were analyzed per mouse) and DCVs per Paneth cells were quantified (n = 6 mice and 20 villus-crypts were analyzed per mouse). (E) NLRP6 expression levels of small intestinal villi from littermate WT and Cnep1r1ΔIEC mice were detected by western blot with anti-NLRP6 antibody. β-Actin was used as a loading control. (F) Relative Nlrp6 expression levels of intestinal crypts from WT and Cnep1r1ΔIEC mice were detected by qRT-PCR. Fold changes were normalized to endogenous 18S (n = 6 mice for each group). (G) Construction diagram showing generation of Nlrp6−/− mice with CRISPR-Cas9. (H) DNA sequencing for Nlrp6 KO validation. (I) Western blot for NLRP6 KO validation with anti-NLRP6 antibody. β-Actin was used as a loading control for western blot. (J) Left: HE staining of duodenum, jejunum, and ileum from littermate WT and Nlrp6−/− mice. Scale bar: 30 μm. Middle: Proportion of normal and abnormal Paneth cells were quantified based on whether Paneth cells displayed a typical staining pattern with distinguishable granules (normal) or disordered, depleted, and/or diffuse staining (abnormal). Right: Numbers of Paneth cells were quantified. n = 6 mice, with 20 selecting maximal crypt sections that displayed all Paneth cells within each crypt were analyzed per mouse. (K) Quantification of Lyz+ puncta per crypt of WT and Nlrp6−/− mice (n = 6 mice for each group). (L) AB-PAS staining of intestines from WT and Nlrp6−/− mice. Scale bar: 35 μm. (M) Left: 3D immunofluorescence staining of ileum crypts from littermate WT and Nlrp6−/− mice. Green: UEA-1, red: EpCAM, and yellow: DCVs’ surface fitted by Imaris software. Scale bar: 35 μm. Right: Volume of DCVs was quantified (n = 6 mice for each group, and 20 crypts were analyzed per mouse). (N) Immunofluorescence staining of organoids from littermate WT and Nlrp6−/− mice. Green: UEA-1, red: EpCAM, and blue: DAPI. Scale bar: 20 μm. (O) Intestinal lumen (left) and tissue-associated (right) bacterial load analysis, quantified by qPCR of 16S rRNA gene copy number in distal ileums (n = 6 mice for each group). (P) qPCR detection of ileal luminal commensal bacteria classified by phylum (n = 6 mice for each group). (Q) Lysozyme in supernatant of stimulated crypts from littermate WT and Nlrp6−/− mice after treatment of DMSO or CCh was measured by ELISA (n = 6 mice for each group). (R) Bacterial load analysis of S. Typhimurium in secreted supernatants. SI crypts of littermate WT and Nlrp6−/− mice were isolated and stimulated by DMSO or CCh. The crypt secreted supernatants were incubated with S. Typhimurium for 30 min, and CFUs were measured (n = 6 mice for each group). Data in D–F, J, K, M, and O–R are shown as means ± SEM; significance was determined by unpaired two-tailed Student’s t test (*P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant). Data in A–F and H–R are representative of at least three independent experiments. Source data are available for this figure: SourceData FS3.

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