Extended Data Fig. 8: IL-17A-induced cytotoxic response in human colon organoids.
From: Somatic inflammatory gene mutations in human ulcerative colitis epithelium
a, Fold-change values of individual sgRNAs in two experiments that used different lines of organoids. sgRNAs are coloured according to their targeting genes, as specified at the top. b, Response of wild-type organoids to treatment with IL-17A in differing growth factor conditions. The organoids showed a cytotoxic response to treatment with IL-17A only in the absence of Noggin. The WNRAIF condition is the medium condition including WNT3A, Noggin, R-spondin-1 (Rspo), A83-01, IGF-1 and FGF-2, but lacking EGF. Data are representative of n = 2 biologically independent experiments with similar results. c, sgRNA targets and confirmation of CRISPR–Cas9-mediated knockout of IL17RA, TRAF3IP2 and NFKBIZ. For knockout of NFKBIZ, three NFKBIZKO lines (#1–3) were generated from different parental organoids. Successful knockout was confirmed by Sanger sequencing in all lines. Loss of IκBζ protein was also confirmed by an immunoassay using an organoid stimulated with IL-17A for 24 h. Result is from a single experiment. d, IL17RAKO and TRAF3IP2KO organoids are resistant to treatment with IL-17A in a Noggin-deficient condition. Right, dots show the relative area of organoids in each well. Results are representative of n = 3 technical replicates. e, NFKBIZKO organoid line 1 tolerates treatment with IL-17A. The other two knockout lines showed a similar response. f, Patient-derived NFKBIZ-mutant organoids (UC02N_IS) are resistant to treatment with IL-17A. Wild-type counterpart organoids (UC02N_UA) were used as a control. Data are representative of n = 2 biologically independent experiments with similar results. g, Patient-derived ZC3H12AD437Y-mutant organoids (UC23N_IS) are tolerant of treatment with IL-17A compared to their wild-type counterparts (UC23N_UA). Data are representative of n = 2 technical replicates with similar results. h, Knockout of TP53 mitigates IL-17A-induced cytotoxicity. Data are representative of n = 2 biologically independent experiments with similar results. Mean + s.e.m (d–h). P values determined by two-sided Welch’s t-test. Scale bars, 1 mm (b, d–h).
