Figure S4.
Apoptosis and pyroptosis alone are not mediating reduction in R-spondin levels in the crypt niche. (A) Representative pictures of IHC of p53 and cleaved caspase 3 in intestines from URI(+/+)Int; p53(Δ/Δ)Int, URI(Δ/Δ)Int; p53(+/+)Int, and URI(Δ/Δ)Int; p53(Δ/Δ)Int mice at indicated time points of tamoxifen treatment. (B and C) Number of p53 (B) and cleaved caspase 3 (C) positive cells per crypt (n = 4) from A. (D) Representative pictures of IHC of URI in intestinal sections from URI(+/+)Int; p53(Δ/Δ)Int, URI(Δ/Δ)Int; p53(+/+)Int, and URI(Δ/Δ)Int; p53(Δ/Δ)Int mice after 6 d of tamoxifen treatment. (E) Representative pictures of TUNEL staining of intestinal sections from URI(Δ/Δ)Int; p53(+/+)Int and URI(Δ/Δ)Int; p53(Δ/Δ)Int mice, following 6 d of tamoxifen treatment. (F) Number of TUNEL positive cells per crypt from E (n = 4). (G) WB of intestinal samples from URI(+/+)Int and URI(Δ/Δ)Int mice after 6 d of tamoxifen treatment. Membranes are blotted with indicated antibodies. (H) Representative pictures of IHC of caspase 1 in intestinal sections from URI(+/+)Int; p53(Δ/Δ)Int, URI(Δ/Δ)Int; p53(+/+)Int, and URI(Δ/Δ)Int; p53(Δ/Δ)Int mice, following 6 d of tamoxifen treatment. Black arrows depict caspase 1 foci in the crypt. (I) Quantification from H. (J) qRT-PCR of Il-1β mRNA levels from URI(+/+)Int, URI(Δ/Δ)Int, URI(+/+)Int; p53(Δ/Δ)Int, and URI(Δ/Δ)Int; p53(Δ/Δ)Int mice, following 6 d of tamoxifen treatment (n = 3, 3, 3, 4 per group). (K) Alkaline phosphatase staining, Alcian blue/PAS staining, IHC of chromogranin A (Chro A), and IF of lysozyme in intestinal sections from URI(+/+)Int; p53(Δ/Δ)Int, URI(Δ/Δ)Int; p53(+/+)Int, and URI(Δ/Δ)Int; p53(Δ/Δ)Int mice, following 6 d of tamoxifen treatment. (J and L–O) Quantification from L of alkaline phosphatase (arbitrary units per villi, A.U./villi) in M, Alcian blue/PAS (number of cells per 100 enterocytes; N), chromogranin A (number of cells per 100 enterocytes; J) and lysozyme (number of positive cells per crypt; O) stainings (n = 4) per group. (P) Scheme depicting Anakinra treatment in URI(+/Δ)Int mice. (Q) qRT-PCR of Rspo1, Rspo2, and Rspo3 mRNA levels of non-treated URI(+/+)Int and URI(+/Δ)Int mice and Anakinra-treated URI(+/Δ)Int mice (n = 3). (R) Representative picture of IHC of MPO positive cells in intestinal tissue from non-treated URI(+/Δ)Int mice and Anakinra-treated URI(+/Δ)Int mice. (S) Quantification of MPO postitive cells from R. Data represent mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001; Student’s t test and one-way ANOVA. Scale bars represent 10 μm in H; 20 μm in D (K, lysozyme panel); 50 μm in E; and 100 μm in A, K, and R. At least 50 crypts and 100 villi were quantified per mouse. IHC and IF are representative of at least three independent mice. WB are representative of at least three independent experiments. Source data are available for this figure: SourceData FS4.

Apoptosis and pyroptosis alone are not mediating reduction in R-spondin levels in the crypt niche. (A) Representative pictures of IHC of p53 and cleaved caspase 3 in intestines from URI(+/+)Int; p53(Δ/Δ)Int, URI(Δ/Δ)Int; p53(+/+)Int, and URI(Δ/Δ)Int; p53(Δ/Δ)Int mice at indicated time points of tamoxifen treatment. (B and C) Number of p53 (B) and cleaved caspase 3 (C) positive cells per crypt (n = 4) from A. (D) Representative pictures of IHC of URI in intestinal sections from URI(+/+)Int; p53(Δ/Δ)Int, URI(Δ/Δ)Int; p53(+/+)Int, and URI(Δ/Δ)Int; p53(Δ/Δ)Int mice after 6 d of tamoxifen treatment. (E) Representative pictures of TUNEL staining of intestinal sections from URI(Δ/Δ)Int; p53(+/+)Int and URI(Δ/Δ)Int; p53(Δ/Δ)Int mice, following 6 d of tamoxifen treatment. (F) Number of TUNEL positive cells per crypt from E (n = 4). (G) WB of intestinal samples from URI(+/+)Int and URI(Δ/Δ)Int mice after 6 d of tamoxifen treatment. Membranes are blotted with indicated antibodies. (H) Representative pictures of IHC of caspase 1 in intestinal sections from URI(+/+)Int; p53(Δ/Δ)Int, URI(Δ/Δ)Int; p53(+/+)Int, and URI(Δ/Δ)Int; p53(Δ/Δ)Int mice, following 6 d of tamoxifen treatment. Black arrows depict caspase 1 foci in the crypt. (I) Quantification from H. (J) qRT-PCR of Il-1β mRNA levels from URI(+/+)Int, URI(Δ/Δ)Int, URI(+/+)Int; p53(Δ/Δ)Int, and URI(Δ/Δ)Int; p53(Δ/Δ)Int mice, following 6 d of tamoxifen treatment (n = 3, 3, 3, 4 per group). (K) Alkaline phosphatase staining, Alcian blue/PAS staining, IHC of chromogranin A (Chro A), and IF of lysozyme in intestinal sections from URI(+/+)Int; p53(Δ/Δ)Int, URI(Δ/Δ)Int; p53(+/+)Int, and URI(Δ/Δ)Int; p53(Δ/Δ)Int mice, following 6 d of tamoxifen treatment. (J and L–O) Quantification from L of alkaline phosphatase (arbitrary units per villi, A.U./villi) in M, Alcian blue/PAS (number of cells per 100 enterocytes; N), chromogranin A (number of cells per 100 enterocytes; J) and lysozyme (number of positive cells per crypt; O) stainings (n = 4) per group. (P) Scheme depicting Anakinra treatment in URI(+/Δ)Int mice. (Q) qRT-PCR of Rspo1, Rspo2, and Rspo3 mRNA levels of non-treated URI(+/+)Int and URI(+/Δ)Int mice and Anakinra-treated URI(+/Δ)Int mice (n = 3). (R) Representative picture of IHC of MPO positive cells in intestinal tissue from non-treated URI(+/Δ)Int mice and Anakinra-treated URI(+/Δ)Int mice. (S) Quantification of MPO postitive cells from R. Data represent mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001; Student’s t test and one-way ANOVA. Scale bars represent 10 μm in H; 20 μm in D (K, lysozyme panel); 50 μm in E; and 100 μm in A, K, and R. At least 50 crypts and 100 villi were quantified per mouse. IHC and IF are representative of at least three independent mice. WB are representative of at least three independent experiments. Source data are available for this figure: SourceData FS4.

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