Fig. 4. ATF-4 overexpression increases H₂S levels via cystathionine gamma-lyase, which is required for longevity.

a Heatmap of gene expression in ATF-4OE (ldIs119), wild type (N2), and atf-4(tm4397) showing genes whose orthologs are directly regulated by mammalian ATF4 (Details are in ‘Methods’, Supplementary Data 4). Absolute levels of expression were compared. Genes in light blue are predicted to be involved in the transsulfuration pathway, which is shown in (b). c ATF-4OE(ldIs119) showed higher cth-2 mRNA levels compared to WT by qRT-PCR. n = 3 independent biological samples in duplicates (each over 200 L4 worms). Mean ± SEM. P values relative to WT determined by one-sample t-test, two-tailed, a hypothetical mean of 1. d Quantification of CTH protein levels in ATF-4OE(ldIs119) compared to WT. n = 6 independent biological trials probed in 3 western blots. One-tailed t-test. e Western blots showing an ATF-4-induced increase in CTH levels was abolished by atf-4 or cth-2 knockdown. NS = non-specific band. f ATF-4 overexpression increases H₂S production capacity in a cth-2-dependent manner. Additional biological trials are shown in Supplementary Fig. 4d. For H₂S quantification, see Supplementary Data 12. g Representative fluorescent microscopy images and quantification showing that H₂S levels in vivo were decreased in either atf-4(tm4397) or cth-2(mg599) mutants compared to WT. Data are represented as Mean ± SEM. n = 3 biological replicates of a total of 15 worms per condition. P values to WT are unpaired t-test, two-tailed. Scale bar = 50 μm. h Lifespan extension induced by ATF-4 overexpression depends upon cth-2. i Lifespan extension induced by ifg-1 knockdown requires cth-2. j Model for how ATF-4 promotes stress resistance and longevity. For statistical details and additional trials in (h) and (i), see Supplementary Data 7. For western blots, source data are provided in Source data File.