Fig. 4.

Midlife Drp1 induction improves mitochondrial respiratory function. a, b Quantification of markers of mitochondrial activity in 28, 37, and 44 day old daGS > UAS-Drp1 females with or without RU486-mediated transgene induction since midlife (day 30 onwards). Complex I activity measurements a in isolated mitochondrial pellet from 28 to 37 day old adult females. n = 5 biological replicates with five flies per replicate; **p < 0.01; two-tailed unpaired t-test. b In situ respirometry of permeabilized muscle bundles from 37 to 44 day old adult females to assess the capacity for oxidative phosphorylation (OXPHOS) and Electron Transport System (ETS) flux, and the flux control ratio of Complex I by rotenone inhibition. n = 6–8 biological replicates with 2 flies per replicate; **p < 0.01 and *p < 0.05; one-way ANOVA/Bonferroni’s multiple comparisons test. c, d Staining of indirect flight muscles c from 37 day old daGS > UAS-Drp1 females with or without RU486-mediated transgene induction from day 30 to day 37, showing mitochondria (green channel, Mitotracker green staining) and levels of superoxide radicals (red channel, staining with MitoSOX reagent). Scale bar is 5 µm. Quantification of free superoxide radicals d; n = 11–16 biological replicates; **p < 0.01; two-tailed unpaired t-test. e qPCR analyses of Hsp60, Hsp10, and mtHsp70 (Hsc70-5) on day 37 in daGS > UAS-Drp1 females with or without RU486-mediated transgene induction from day 30 to day 37. n = 5 biological replicates with 3 flies per replicate; p > 0.05 and is non-significant (n.s.); two-tailed unpaired t-test. Bars a depict mean ± s.d. Boxplots b, d and e display the first and third quartile, with the horizontal bar at the median and whiskers showing the most extreme data point, which is no more than 1.5 times the interquartile range from the box. RU486 was provided in the media at a concentration of 25 μg/ml