Fig. 6. Hypusination of eIF5A acts downstream of spermidine to mediate IF-associated longevity.

a, Scheme of the hypusination pathway. DHS, deoxyhypusine synthase; DOHH, deoxyhypusine hydroxylase; eIF5A, eukaryotic translation initiation factor 5A-1. b, Representative immunoblot of hypusinated eIF5A (eIF5A^H) levels in yeast GFP-Atg8 WT and ∆spe1 after 6 h of −N. c, Quantification of b. n = 8(WT), 7(∆spe1) biologically independent samples (yeast cultures). d, Representative maximum projection images of confocal microscopy images of female w¹¹¹⁸ fly central brain regions probed for eIF5A and hypusine by immunofluorescence. Before dissection, the flies were fasted for 0 h (ad lib) or 12 h, starting at 20:00. Scale bar, 50 µm. e, Quantification of signal intensities in d. n = 22 (ad lib), 23 (fasted) fly brains. f, Immunoblots of 48 h fasted C. elegans assessed for hypusine and GAPDH. g, Quantification of f. n = 4 biologically independent samples (worm lysates). h, Representative immunoblot of eIF5A^H, eIF5A and GAPDH signals of liver samples from ad lib and fasted (14–16 h) young, male C57BL/6 mice. i, Quantification of h. n = 7 mice. j, eIF5A^H and total eIF5A levels in human PBMCs after increasing fasting times, measured by capillary immunoblotting. RF, 3 days after re-introduction of food. n = 17 (days 0, 1 and 3), 15 (day 5), 16 (day 7 and RF) volunteers. k, Representative immunoblot of yeast Lia1-6xHA, assessed for HA-tags and GAPDH after 6 and 24 h −N. l, Quantification of Lia1-6xHA levels as depicted in k. n = 8 biologically independent samples (yeast cultures). m, Quantification of relative mRNA expression of dhps-1, dohh-1 and iff-1 in 24 h fasted C. elegans. n = 3 (iff-1), 5 (dhps-1 and dohh-1) biologically independent experiments. n, Quantification of relative mRNA expression of DHPS, DOHH and EIF5A in 6 h starved U2OS cells. n = 3 (DOHH), 4 (DHPS and EIF5A) biologically independent experiments. o, PI-negative (live) yeast cells during chronological lifespan analysis of WT and ∆lia1 yeast in control and −N medium. n = 12 biologically independent samples (yeast cultures). p, Lifespan of C. elegans N2 fed control (CTL) or dhps-1(RNAi) (homologue of DHS) expressing bacteria during IF^48:48. Note that the experiments and statistics were performed together with the groups depicted in Fig. 4g,i. n = 913 (CTL ad lib), 750 (CTL IF), 862 (dhps-1(RNAi) ad lib), 776 (dhps-1(RNAi) IF) worms. Statistics used were two-way ANOVA with Holm-Šídák’s multiple comparisons test (c,j,l,o), two-tailed Student’s t-tests with Holm-Šídák’s multiple comparisons test (e,i,m,n), two-tailed Student’s t-test (h) and log-rank test with Bonferroni correction (p). Bar and line graphs show the mean ± s.e.m. Source numerical data and unprocessed blots are available in source data.

Source data