Fig. 1: Mitochondrial protein import inhibition elicits morphological and bioenergetic adaptations of the mitochondrial network and regulates lifespan.

a, b Transgenic animals expressing a mitochondrial-targeted GFP in the intestine (a) or body-wall muscles (b), were grown from hatching until day 1 of adulthood on the indicated RNAi expressing bacteria and then monitored by confocal microscopy. n = 3 biologically independent experiments with similar results. c Epifluorescence images depicting total COX-4 endogenous protein levels in animals grown from hatching until day 1 of adulthood on the indicated RNAi expressing bacteria (left panel) and COX-4::GFP fluorescence quantification in violin plot (right panel). n = 3 biologically independent experiments with at least 20 worms per condition. Exact sample size and P values are included in Source Data file. d–f Day 1 adult animals were stained with Mito Tracker Green, which labels mitochondria independently of membrane potential (d), TMRE, which is a membrane potential-dependent mitochondrial dye (e), and Mito Tracker Red CM-H2Xros, which is a mitochondrial ROS specific dye (f). The violin plots represent the mean fluorescent intensity of at least 20 worms. All treatments were compared to control with two-tailed t-test. n = 3 biologically independent experiments with at least 20 worms. Exact sample size and P values are included in the Source Data file. g Immunoblot analysis of total ATP-1 protein levels in animals grown from hatching until day 1 of adulthood on the indicated RNAi expressing bacteria. n = 2 biologically independent experiments. h–k Lifespan curves of control-treated and animals treated with timm-23(RNAi) (h), tomm-40(RNAi) (i), timm-22(RNAi) (j), and gop-3(RNAi) (k) from egg. n = 3 biologically independent experiments. l Lifespan curve of DECA-treated animals in various concentrations from the egg. n = 2 biologically independent experiments. Statistical analysis for lifespan curves was performed with the Log-rank (Mantel–Cox) test; detailed values are shown in Supplementary Table 2. m Quantification of COX-4::GFP fluorescence upon treatment with different DECA concentrations (depicted in violin plot). n Quantification of mitochondrial membrane Δψ (TMRE staining) upon treatment with different DECA concentrations. o Violin plot of quantified Mitotracker Green staining upon treatment with different DECA concentrations. m–o n = 2 biologically independent experiments. All treatments were compared to control with two-tailed t-test (**** denotes P < 0.0001, *** denotes P < 0.001, ** denotes P < 0.01 and * denotes P < 0.05) Exact sample size and P values are included in Source Data file. a.u. arbitrary units.