Fig. 6. GSH restriction upregulates mitochondrial ROS signaling in C. elegans.

a Genes induced by acivicin and by life-extending mitochondrial mutations overlap significantly. Numbers represent the expression log₂ fold change compared to wt or untreated worms. See full list of differentially regulated genes in Supplementary Data 2. Data for gene expression in daf-2, clk-1, isp-1 and nuo-6 are from³². Up- and down-regulated genes are indicated, respectively, in red and blue. Genes which are regulated in daf-2 worms are highlighted in gray. *-genes regulated by Hg²⁺, CH₃HgCl or Cd²⁺. Statistical significance for the overlap between sets upregulated more than two folds by: acivicin (34 genes) and nuo-6 (1285 genes) – 22 genes overlap, RF = 10.3, p value = 7.93e−19; acivicin and clk-1 (328 genes) – 13 genes overlap, RF = 23.9, p value = 2.48e−15; acivicin and isp-1 (609 genes) – 19 genes overlap, RF = 18.8, p value = 9.01e−21. RF – representation factor is the number of overlapping genes divided by the expected number of overlapping genes and p is normal approximation of hypergeometric probability. b, c Representative fluorescent images (left panels) and quantifications (right panels) demonstrating the tbb-6 counter-regulation by acivicin (n = 70) and NAC (n = 30-55) (b) and the tbb-6 induction by acetaminophen (AAPh, 20 mM, n = 35), acivicin (75 µM, n = 35), dinitrophenol (DNP, 100 µM, n = 35), and cadmium (Cd²⁺, 50 µM, n = 65) (c). L4 stage worms were transferred to plates supplemented with chemicals as indicated, and incubated for 24 h at 20 °C before imaging. n = 30–70 worms over three independent experiments. See also Supplementary Tables 8 and 9. d Representative fluorescent and bright light images (left panels) and quantifications (right panel) demonstrating mitochondrial ROS staining by MitoTracker CM-H₂X in control and acivicin-treated worms (n = 79). Worms developed till stage L3 on control and acivicin supplemented plates. See also Supplementary Table 10. In b, c and d the box plots indicate median (middle line), 25th, 75th percentile (box) and 5th and 95th percentile (whiskers) as well as maximum, minimum and mean (single points). In all graphs p values are: n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; two-tailed t-tests.