Extended Data Fig. 3: Mice with fibroblast-specific ablation or fibroblast-restricted expression of Cox-2.

a, Immunofluorescence of ileum and colon sections from Col6-cre-Rosa26^tdTomato/+ mice (scale bar, 20 μm) and of a small intestinal tumour section from an Apc^Min/+-Col6-cre-Rosa26^tdTomato/+ mouse (scale bar, 150 μm). Data are representative of two experiments. b, Efficiency of Col6-cre-mediated recombination of a lox-stop-lox tdTomato reporter in Pdgfra^high and Pdgfra^low Cd45⁻ cells determined by flow cytometry in intestinal mesenchymal and lamina propria cells isolated from the small intestine and the colon of Col6-cre-Rosa26^tdTomato/+Pdgfra^eGFP/+ mice in one experiment. c, Ptgs2 relative gene expression (RE) in whole tissue, isolated IECs, FACS-sorted Col6Cre⁺ fibroblasts (CD45⁻tdTomato⁺) and Col6Cre⁻ mesenchymal cells (CD45⁻tdTomato⁻) from the small intestine of Col6-cre-Rosa26^tdTomato/+ mice (n = 3, pooled). Representative of two experiments. d, Efficiency of Col6-cre-mediated Ptgs2 gene ablation in Col6-Cre⁺ mesenchymal cells determined by RT–qPCR analysis of Ptgs2 expression in FACS-sorted Col6-cre⁺ fibroblasts (eGFP⁺) from the small intestine of Col6-cre-Rosa26^mT/mGPtgs2^f/+ (n = 3) and Col6-cre-Rosa26^mT/mGPtgs2^f/f (n = 3) mice. Unpaired two-tailed Welch's t-test. e, Expression of the Ptgs2 gene in whole tissue ileum of littermate Ptgs2^f/f and Ptgs2^ΔFibr mice (n = 7 each). Two-tailed t-test. f, Spleen weight of 5.5-month-old Apc^Min/+Ptgs2^f/f (n = 8) and Apc^Min/+Ptgs2^ΔFibr (n = 6) mice. Average spleen weight of (n = 6) normal littermates (Ptgs2^f/f) is displayed for comparison. Two-tailed t-test. g, Survival analysis of Apc^Min/+Ptgs2^f/f (n = 12) and Apc^Min/+Ptgs2^ΔFibr (n = 12) mice. A two-tailed P = 0.00009687 was calculated by log-rank test. h, Size of 274 adenomas from 5.5-month-old Apc^Min/+Ptgs2^f/f (n = 16) and Apc^Min/+Ptgs2^ΔFibr (n = 18) mice. The whiskers extend from minimum to maximum and the box extends from the 25th to 75th percentiles with the median indicated. Two-tailed Mann–Whitney test. i, Generation of knockin mice bearing a lox-stop-lox cassette insertion in intron-3 of the Ptgs2 gene which prevents its expression (Ptgs2^OFF). Col6-cre-mediated excision of the lox-stop-lox cassette reactivates Ptgs2 expression specifically in fibroblasts (Ptgs2^FibrON). The orange box depicts an frt site remaining from the flp-mediated removal of an frt-flanked PGK-neomycin selection cassette (see Methods). j, Ptgs2^f/f (n = 30) and Ptgs2^ΔFibr (n = 24) mice were subjected to 10 weekly intraperitoneal injections with 10 mg kg⁻¹ azoxymethane as displayed. Quantification of the number of dysplastic foci and microadenomas per mouse and quantification of tumour size is shown. Statistical significance was tested by two-tailed Mann–Whitney test. k, Quantification of intestinal epithelial populations in the ileum of littermate Ptgs2^f/f and Ptgs2^ΔFibr mice (n = 3–5 per genotype). Immunostaining was performed for markers of Paneth cells (lysozyme), tuft cells (Dclk1), enteroendocrine cells (chromogranin A) and stem cells (Olfm4). Goblet cells were identified by periodic acid Schiff (PAS) staining and enterocytes were identified by detecting alkaline phosphatase enzymatic activity. Incorporation and immunohistochemical detection of BrdU was used to determine the numbers of cycling cells. Data for each mouse represent mean number of positive cells per crypt or crypt–villus unit as indicated. N = 400–822 crypts and/or villi were evaluated per staining. Statistical comparisons were performed with two-tailed unpaired t-test except for Olfm4⁺ cells for which unpaired t-test with Welch’s correction was applied. Scale bars, 50 μm. All data represent mean ± s.e.m. unless otherwise indicated. ns, non-significant; *P < 0.05, **P < 0.01, ***P < 0.001.

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