Fig. 3: ALKB-1 inactivation-induces PME delay, mediated by UPR^mt activation and mtDNA amplification.

Representative bright-field and GFP fluorescence images (a) and quantification (b) of the hsp-6p::gfp reporter in wild-type and alkb-1^D247A males treated with control or atfs-1 RNAi (n = 30 per group), with scale bars of 100 μm. The experiment was performed three times with similar results. c, e Representative DIC and MTR-stained paternal mitochondrial images of the 32- and 64-cell stage cross-fertilized embryos from the indicated crosses with scale bars of 10 μm. d, f Quantification of the number of MTR-labeled mitochondrial clusters in (c, e), respectively. In (d), n = 19 (N2, 32-cell), n = 15 (N2, 64-cell), n = 23 (alkb-1^D247A, 32-cell), n = 19 (alkb-1^D247A, 64-cell), n = 22 (alkb-1^D247A;atfs-1, 32-cell), n = 18 (alkb-1^D247A;atfs-1, 64-cell), n = 22 (alkb-1^D247A;atfs-1 males × alkb-1^D247A hermaphrodites, 32-cell), n = 14 (alkb-1^D247A;atfs-1 males × alkb-1^D247A hermaphrodites, 64-cell), n = 21 (alkb-1^D247A males × alkb-1^D247A;atfs-1 hermaphrodites, 32-cell), n = 17 (alkb-1^D247A males × alkb-1^D247A;atfs-1 hermaphrodites, 64-cell). In (f), n = 16 (N2, 32-cell), n = 15 (N2, 64-cell), n = 15 (alkb-1^D247A, 32- and 64-cell), n = 14 (alkb-1^D247A;dve-1, 32- and 64-cell), n = 14 (alkb-1^D247A;lin-65, 32-cell), n = 13 (alkb-1^D247A;lin-65, 64-cell), n = 13 (alkb-1^D247A;ubl-5, 32- and 64-cell). g Schematic diagram illustrating the relationship among LONP-1, mitochondrial ATFS-1 accumulation, and mtDNA replication. h Western blot analyses showing the accumulation of mitochondrial ATFS-1 in atfs-1p::atfs-1::TY1::egfp::3×flag worms treated with control, alkb-1, cco-1, and lonp-1 RNAi. cco-1 and lonp-1 RNAi served as the positive control, and N2 worms were used as the negative control. n = 3 biological repeats. i Quantification of the levels of total mtDNA in both N2 and alkb-1^D247A, including males and early embryos (eggs) (n  =  9 biologically independent samples). j Quantification of the levels of total mtDNA in males of both N2 and alkb-1^D247A treated with control or atfs-1 RNAi (n  =  9 biologically independent samples). In (d, f), embryos were derived from 3 to 5 independent biological crosses per condition. The experiment was performed twice with similar results. Data were represented as mean ± SD. p-values were determined by Ordinary one-way ANOVA with Tukey’s multiple comparisons test (b, d, f, j), and unpaired two-tailed t-test (i). Source data are provided as a Source data file.