Extended Data Fig. 7: Notum regulates intestinal stem cell function.
From: Notum produced by Paneth cells attenuates regeneration of aged intestinal epithelium
a, Notum gene targeting. Schematic represents sites of genome editing. Gene editing was confirmed by PCR with primers flanking the editing site (174 bp product for Notum KO1 and 294 bp product for Notum KO2) and hitting the edited site (84 bp product for Notum KO1 and 188 bp product for Notum KO2). Representative agarose gel images from two independent experiments with similar results are shown. b, Regenerative growth of Notum knockout organoids. De novo crypt domains were quantified two days after subculture (n = 5 repeated experiments with the same organoid lines). Representative images of organoids two days after subculture are shown. Scale bar, 100 μm. c, Schematic presenting in vivo competition assay of gene-edited organoid growth by orthotopic transplantation to immunodeficient Rag2−/− mice. Representative colonoscopy, necroscopy and histology images used for assay quantification (n = 8 mice transplanted). Scale bars, 1 mm for necroscopy and 200 μm for histology. d, Representative images of CRISPR-targeted young and old organoids two days after subculturing (n = 4 mice per group). Scale bar, 100 μm. e, Relative Notum expression in organoids with synergistic activator mediator complex (SAM) targeted to Notum promoter (dA Notum) grown for two days in ENR medium. Three independent experiments; relative to control (dA Tom). f, Quantification and representative images of day-5 colonies formed by isolated CD24medSSClo cells from Notum activator (dA Notum) and control (dA Tom) organoids. Scale bar, 100 μm. n = 4 repeated experiments with the same organoid line. g, RT–qPCR analysis of relative Axin2 and Lgr5 expression in CD24medSSClo cells sorted from Notum activator (dA Notum) organoids. Values show fold change (expressed in log2) in comparison to control (dATom) (n = 3 replicate wells per organoid line). 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.e.m.; two-tailed unpaired Student’s t-test; exact P values shown in corresponding panels. For gel source data see Supplementary Fig. 3.
