Extended Data Fig. 8: Notum inhibitor ABC99 prevents Wnt inactivation.
From: Notum produced by Paneth cells attenuates regeneration of aged intestinal epithelium
a, Flow cytometry analysis of cell populations in primary organoids treated for eight days with 500 nM ABC99 (n = 3 mice) relative to DMSO control. Student’s paired t-test. b, Clonogenic growth of Lgr5hi stem cells on day 5 treated with or without 50 nM ABC99 and/or 500 ng ml−1 recombinant Notum (two independent experiments with similar results, one experiment with three replicate wells shown). c, Relative weight of mice treated with daily injections of ABC99 (10 mg per kg (body weight)) or control (vehicle or ABC101 10 mg per kg (body weight)) (n = 10 mice for young control and young ABC99, n = 8 mice for old control and n = 9 mice for old ABC99). Daily data points represent median (circles) and interquartile range (dashed line). d, Clonogenic growth of young Lgr5hi stem cells co-cultured with young or old Paneth cells from mice treated with ABC99 or control (n values for analysed mice shown). Combinations compared to average of co-cultures with young control (−) and old ABC-treated (+) Paneth cells. Control mice received an equal amount of the inactive analogue ABC101 (yellow circles) or vehicle. e, Representative image of immunofluorescent staining of ileal crypts used for quantification of nuclear β-catenin (white) intensity. Paneth cells (red arrowheads) and CBCs (green arrowheads) were identified by cellular and nuclear (DAPI, blue) morphology. Their nuclear β-catenin levels were compared to transit-amplifying cells (white arrowheads). Scale bar, 20 μm. Experiment was repeated twice with a total of 26 mice all showing strongest nuclear β-catenin at the crypt bottom. f, Immunofluorescent staining of histological sections from old ileum. β-catenin (white), lysozyme (red) and DAPI (nuclei, blue). Scale bar, 10 μm. Quantification of relative nuclear β-catenin intensity of Paneth cells (red arrowhead) (n values for analysed mice shown). For quantification of CBCs (green arrowhead) see Fig. 3d. g, Immunofluorescent staining of histological sections from old ileum. Olfm4, green; EdU, red; DAPI (nuclei), blue. Scale bar, 20 μm. Quantification of EdU+ cellular frequencies within the crypt (n values for analysed mice shown). Y, mice between 3 and 9 months of age; O, mice over 24 months of age in all experiments. In box plots, unless otherwise indicated, the line represents median, the box shows interquartile range and whiskers represent the range. All other data are mean ± s.d.; two-tailed unpaired Student’s t-test; exact P values shown in corresponding panels.
