Fig. 1: Identifying proteome dynamics during regeneration of young small intestines.

a, A schematic of 5-FU induced regeneration in young mice. i.p., intraperitoneal. b, The relative body weight of young mice treated with a single dose of 5-FU or PBS as control. The body weight of each mouse was normalized to its body weight at the time of injection. n = 18 PBS, n = 17 5-FU mice. The red and blue lines indicate the median body weight for each treatment group on each day. The P values were calculated using the two-tailed Welch’s t-test by comparing the body weights of 5-FU-treated mice with PBS-treated controls on the indicated day and by two-way ANOVA for overall day and treatment comparisons. c, The representative pictures of haematoxylin and eosin (H&E) staining of the small intestine from indicated treatments and timepoints. Scale bars, 50 µm. d, The quantification of the number of crypts per millimetre of small intestine in the indicated groups. The PBS-treated mice from different days were combined. Each dot represents one mouse. n = 12 PBS, n = 4 5-FU mice per group. The data are presented as mean ± s.d. The P value was calculated using the two-tailed Welch’s t-test. e, The quantification of the number of cells per micrometre of villus in the indicated groups. PBS-treated mice from different days were combined. Each dot represents one mouse. n = 11 PBS, n = 4 5-FU mice per group. The data are presented as mean ± s.d. The P value was calculated using the two-tailed Welch’s t-test. f, Left: the representative pictures of pH3 staining from indicated treatment and timepoint. Scale bar, 100 µm. The asterisks indicate the pH3⁺ crypts. Right: the percentage of pH3⁺ crypts in the indicated groups. The PBS-treated mice from different days were combined. Each dot represents one mouse. n = 14 PBS; n = 5 day 2, n = 6 day 5, n = 11 day 7 5-FU. The data are presented as mean ± s.d. The P value was calculated using the two-tailed Welch’s t-test. g, The classification of protein groups quantified by proteomics according to their abundance changes relative to the PBS controls. h, The abundance profiles for the four clusters of proteins affected by 5-FU. The representative REACTOME gene sets significantly enriched in each cluster are shown. Stand., standardized; SLC, solute carrier; ENOS, endothelial nitric oxide synthase. i, The abundance profile of ribosome (67 proteins) and MCM (6 proteins) complexes. The protein abundances of individual complex members were normalized to the median complex abundance of PBS-treated mice, and profiles plotted using a locally estimated scatterplot smoothing function. The shaded area around the regression line represents the 95% confidence interval. n = 16 PBS, n = 5 day 2, n = 5 day 5, n = 4 day 7 5-FU. j, A scheme of puromycin incorporation assay performed on freshly isolated crypts. k, Left: a representative immunoblot for puromycin incorporation. Right: the quantification of puromycin incorporation relative to Ponceau staining (loading control). Each dot represents one mouse. n = 3 mice per group. The data are presented as mean ± s.d. The P value was calculated using the two-tailed Welch’s t-test. Panels a and j created with BioRender.com.

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