Fig. 6: Mitophagy but not DAF-16 mediates the effects of MI.

a daf-16-NL could not block the longevity effect of MI, N = 2. b The longevity effect of MI could be largely blocked by pink-1-NL (ok3538), N = 5. c pink-1-NL could block the mobility promoting effect of MI seen in control N2 worms (n = 10 in each group, *p < 0.05), N = 3. d Immunoblotting result of PINK1 in differentiated C2C12 cell samples, N = 3. e Quantification of the immunoblotting result (Data are presented as mean values ± SEM, two-sided t-test **p < 0.004). f Representative images show MI supplementation decreased SQST-1::GFP aggregates in tail muscles of AD_10 worms (bpIs287[Pmyo-3::sqst-1::gfp]), scale bar 10 μm, N = 2. g Quantification of Fig. 6f like worms, one worm tail region per image, under 63x CONFOCAL microscopy (Data are presented as mean values ± SEM, n = 26, two-sided t-test *p = 0.005). h DA2123 (adIs2122 [lgg-1p::GFP::lgg-1 + rol-6(su1006)]) worms were treated for 8 days from AD_1. The figure shows MI induced co-localization of the autophagy marker (GFP::LGG-1) and mitochondria (stained with mito-tracker), scale bar 20 μm for full-size image, scale bar 2 μm for the zoom-in image, N = 2. i Quantification of Fig. 6h using the Colocalization Index (Methods). M1 is to quantify the overlapping signal over the mito-tracker signal, and M2 is to quantify the overlapping signal over GFP::LGG-1 signal. MI stands for MI 50 mM. Data are presented as mean values ± SEM, n = 13, p values were determined by a two-sided t-test. j Mitochondrial respirometry shows MI increased the mitochondrial activity in the worm, left and right represent basal and maximal oxygen respiration rate (OCR) respectively (Data are presented as mean values ±  SEM, two-sided t-test), N = 2. k Mitochondrial respirometry shows MI increased the mitochondrial activity in differentiated C2C12 cells, N = 2. l A schematic model summarizes the aging-alleviating effects of MI supplement. The anti-aging effect of MI is through converting to PI(4,5)P₂ and activating PTEN and mitophagy downstream, but not through the classical PTEN downstream insulin IGF-1 pathway components. Source data are provided as a Source Data file.