Fig. 2: NOX1-derived ROS regulates the balance of self-renewal and differentiation in the distal colon in homeostasis and during regeneration.

a–c H&E staining images (a), quantification of crypt depths (b) and cell number per crypt (c) of distal colons from WT and NOX1^KO littermates under baseline. Each dot indicates one ROI in (b) (n = 3 mice, 4 or 5 ROI/mouse) and one crypt in (c) (n = 3 mice, 14 or 15 crypts/mouse). d, e Flow cytometry plot (d) and quantification (e) of BrdU incorporation in intestinal epithelial cells (Live/dead⁻CD45⁻EpCam⁺) from distal colons of WT and NOX1^KO littermates under baseline. Each dot indicates one mouse (n = 6 mice). f–h Representative fluorescence images (g) and quantifications (f) (n = 4 mice, 2 or 3 ROI/mouse) of Nox1 mRNA intensity, quantifications (h) of Ki67⁺ cells per crypt of distal colons from WT and NOX1^KO littermates under fasting and refeeding. Each dot indicates one ROI (n = 7 mice in WT group, n = 5 in NOX1^KO group, 8 ROI/mouse). i–k Bright field images (i), colonoids formation numbers (j) and surface area (k) of colonoids generated from isolated crypts in distal colons of WT and NOX1^KO littermates, treated with or without NAC or diamide separately. Each dot indicates one replicate in (j) and one organoid in (k) (n = 3 mice per group). l Percentage stem colonoids (spheroid) in WT and NOX1^KO three days after plating and in WT following incubation with catalase (n = 4 mice). m Percentage stem colonoids (spheroid) in WT and NOX1^KO three days after plating and in NOX^KO after addition of glucose oxidase and glucose (n = 4 mice). Experiments were performed no fewer than 2 times. Data are presented as mean ± SEM. Statistical significance was determined using two-tailed unpaired Student’s t tests in (b), (c) and (e), one-way ANOVA in (f), (l) and (m), Two-way ANOVA in (h), (j) and (k); ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.