Fig. 2: Inhibition of hypusination of eIF5A arrests colonic organoid growth.

a, b Isotope ratio of putrescine and spermidine in colon organoids cultured with isotope-labelled ¹⁵N₂ putrescine were analysed via GC-MS. The black bar indicates the percent putrescine (PUT) or spermidine (SPD) of ¹⁵N₂ putrescine or ¹⁵N₂ spermidine enrichment in total corresponding polyamines, respectively (n = 3, independent cultures). c Polyamine synthesis pathway and eIF5A hypusination. Hypusine is formed by conjugation of the aminobutyl moiety (green) of spermidine to a lysine residue. MCHA and GC7 inhibit SPDS and DHS, respectively. SPMS: spermine synthase, DOHH: deoxyhypusine hydrogenase. d Representative microscopy images of organoid cultured with or without MCHA and spermidine (SPD). Scale bars: 200 µm. The average size of each organoid was quantified using Image J software (n = 4, independent cultures). e Mki67 mRNA expression was analysed by q-PCR (GF: n = 8, WT: n = 6, SK930: n = 8, independent animals). Western blot analysis of hyp-eIF5A in f organoids treated with MCHA (n = 3) and in g CECs of gnotobiotic mice (n = 5). h Representative microscopy images of organoids cultured with or without GC7. Scale bars: 200 µm. Reproducibility of results was confirmed by performing two independent experiments. i Western blot analysis of hyp-eIF5A in organoids treated with MCHA (n = 3, independent cultures). j Western blot analysis of hyp-eIF5A in CECs of SPF mice with GC7 treatment (n = 3, independent animals). k Representative microscopic images of EdU assay and quantification of EdU-positive cells/crypt in control or GC7 treated mice. EdU-positive cells (green). Nuclei are counterstained with DAPI (blue). Scale bars: 100 µm. Three randomly selected areas were examined per slide (n = 3, independent animals). All data shown represent the mean ± SEM. Statistical significance was calculated using d, f the one-way ANOVA followed by Tukey’s post-hoc test, e, i Welch’s ANOVA followed by Dunnett’s T3 post-hoc test and g, j, k Student’s t-test (two-sided).