daf-2 mutation or daf-2 knockdown does not enhance mitochondrial import. (a and b) glp-1(e2141ts) animals were grown at 25°C on bacteria expressing daf-2 dsRNA (diluted to 1:1 ratio with bacteria containing the empty RNAi vector alone) from hatching until the first day of adulthood. Mitochondria were isolated on day 1 of adulthood and subjected to import assay followed by Western blot analysis. Graph is presented as mean ± SD, n = 3. (c and d) CF512 fer-15(b26) II; fem-1(hc17ts) I and CF596 daf-2(mu150) III; fer-15(b26); fem-1(hc17ts) worms were grown at 20°C until larval stage L2 and then transferred to 25°C. Mitochondria were isolated on day 1 of adulthood and subjected to import assay followed by Western blot analysis. Import efficiency was quantified by measuring the mature imported protein as detected by the DHFR antibody, followed by analysis with unpaired Student’s t test (two-tailed; b and d). Graph is presented as mean ± SD, n = 3. Arrowheads: mature (imported) DHFR with the MTS cleaved off. (e)glp-1(e2141ts) animals were grown at 25°C on bacteria expressing daf-2 dsRNA (diluted to 1:1 ratio with bacteria containing the empty RNAi vector alone) from hatching until the first of adulthood. RNA was isolated on day 1 of adulthood, and qPCR analysis was performed. Expression was normalized against three housekeeping genes and quantified with Student’s t test (two-tailed). The graph is presented as mean ± SD, n = 4. Source data are available for this figure: SourceData FS4.