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. Author manuscript; available in PMC: 2022 Dec 1.
Published in final edited form as: Nat Aging. 2022 May 16;2(6):494–507. doi: 10.1038/s43587-022-00214-y

Figure 6. Neuronal BNIP3 induction requires autophagy to delay systemic aging.

Figure 6.

(a) Immunostaining of indirect flight muscles from young (10-day-old) and aged (30-day-old) elavGS>UAS-Atg1RNAi,UAS-BNIP3 flies with or without RU486-mediated transgene induction from day 5 onward, showing mitochondrial morphology (green channel, anti-ATP5a), rhodamine phalloidin (RP, magenta channel), and nuclear DNA (blue channel, stained with DAPI). Scale bar is 10 μm.

(b) Quantification of mitochondrial size in muscle as shown in (a). n = 8 biologically independent animals per condition. *p=0.0400, **p=0.0011, non-significant (n.s.); Kruskal-Wallis test/Dunn’s multiple comparisons test.

(c) Staining of indirect flight muscles from young (10-day-old) and aged (51-day-old) elavGS>UAS-BNIP3 flies with or without RU486-mediated transgene induction from day 5 onward, showing TMRE fluorescence. Scale bar is 10 μm.

(d) Quantification of mitochondrial membrane potential measured by TMRE staining as shown in (c). n = 9 young, 12 aged RU−, and 10 aged RU+ biologically independent animals. *p=0.0281, **p=0.0018, non-significant (n.s.); Kruskal-Wallis test/Dunn’s multiple comparisons test.

(e) Immunostaining of guts from young (10-day-old) and aged (51-day-old) elavGS>UAS-Atg1RNAi,UAS-BNIP3 flies with or without RU486-mediated transgene induction from day 5 onward, showing intestinal stem cell proliferation (red channel, anti-PH3, white arrows) and nuclear DNA (blue channel, stained with DAPI). Scale bar is 50 μm.

(f) Quantification of total number of PH3+ cells in gut as shown in (e). n = 12 young, 11 aged RU−, and 8 aged RU+ biologically independent animals, as indicated **p=0.0011, ***p<0.0001, non-significant (n.s.); Kruskal-Wallis test/Dunn’s multiple comparisons test.

(g) Immunostaining of guts from young (10-day-old) and aged (51-day-old) elavGS>UAS-Atg1RNAi,UAS-BNIP3 flies with or without RU486-mediated transgene induction from day 5 onward, showing mitochondrial morphology (green channel, anti-ATP5a) and nuclear DNA (blue channel, stained with DAPI). Scale bar is 5 μm.

(h) Quantification of mitochondrial area in gut as shown in (g). n = 8 biologically independent animals per condition. **p=0.0014 (young vs. aged RU−), **p=0.0044 (young vs. aged RU+); Kruskal-Wallis test/Dunn’s multiple comparisons test.

(i) Intestinal integrity during aging of elavGS>UAS-Atg1RNAi,UAS-BNIP3 flies with or without RU486-mediated transgene induction from day 5 onward. n = 5 vials with 30 biologically independent animals per vial on day 10. n.s. = non-significant; two-way ANOVA/Šídák’s multiple comparisons test. RU486 or vehicle was provided in the media at a concentration of 5 μg/ml in the indicated treatment groups. Data are presented as scatter plots overlaying mean values +/− SEM.