Fig. 3: The cell-intrinsic function of NOX1 regulates ISC self-renewal and cell fate in the distal colon.

a Quantification of Lgr5 mRNA intensity in Supplementary Fig. 4a from freshly frozen distal colons. Each dot indicates one ROI (n = 3 mice, 2 or 3 ROI/mouse). b Proportions of Lgr5-EGFP⁺ cells (gated from Live/dead⁻CD45⁻EpCam⁺) from distal colons of WT; Lgr5^creERT2+ and NOX1^KO; Lgr5^creERT2+ naïve mice. Each dot indicates one mouse (n = 5 mice). c, d Flow cytometry plot (c) and proportions (d) of BrdU⁺ Lgr5-EGFP⁺ cells (gated from Live/dead⁻CD45⁻EpCam⁺Lgr5-EGFP⁺) from distal colons of WT; Lgr5^creERT2+ and NOX1^KO; Lgr5^creERT2+ mice under fasting and refeeding. Each dot indicates one mouse (n = 5 mice). e, f Bright field images (e) and quantifications (f) of primary organoid formation per 1000 sorted Lgr5-EGFP⁺ cells of distal colons from either WT; Lgr5^creERT2+ or NOX1^KO; Lgr5^creERT2+ mice. Each dot indicates one technical replicate (n = 2 mice). g Generation of lineage tracing mice WT; Lgr5^tdTomato and NOX1^KO; Lgr5^tdTomato mice (left) and representative fluorescence images of freshly frozen distal colon sections (right), immunolabelled for tdTomato and EGFP (n = 3 mice). h, i Flow cytometry plot (h) and quantification (i) of total tdTomato⁺ cells (gated from Live/dead⁻CD45⁻EpCam⁺) from distal colons of (g) mice. Each dot indicates one mouse (n = 6 mice). j, k Flow cytometry plot (j) and quantification (k) of tdTomato⁺Lgr5-EGFP⁺ cells (gated from Live/dead⁻CD45⁻EpCam⁺ Lgr5-EGFP⁺) from distal colons of (g) mice. Each dot indicates one mouse (n = 6 mice). Experiments were performed no less than 2 times. Data are presented as mean ± SEM. Statistical significance was determined using two-tailed unpaired Student’s t tests in (a), (b), (f), (i) and (k), Two-way ANOVA in (d); ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.