Extended Data Figure 2. Related to Figure 1. Neuronal BNIP3 induction prevents loss of neurons in aged brains.
(a) TOPRO staining of nuclei in brains from young (10-day-old) and aged (30-day-old) elavGS>UAS-BNIP3 flies with or without RU486-mediated transgene induction from day 5 onward. Scale bar is 5 μm.
(b) Quantification of nuclei per 350 μm2 of optic lobe as shown in (a). n = 12 young, 12 aged RU−, and 15 aged RU− biologically independent animals per condition, as indicated. *p=0.0219, **p=0.0097; one-way ANOVA/Tukey’s multiple comparisons test.
(c) Immunostaining of brains from young (10-day-old) and aged (30-day-old) elavGS>UAS-BNIP3 flies with or without RU486-mediated transgene induction from day 5 onward, showing cleaved (activated) caspase-3 (green channel) and nuclear DNA (blue channel, stained with To-Pro-3). Scale bar is 5 μm.
(d) Quantification of cleaved caspase-3 in one optic lobe per fly as shown in (a). n = 8 young, 9 aged RU−, and 10 aged RU+ biologically independent animals per condition, as indicated. *p=0.0140 (young vs. aged RU−), *p=0.0119 (aged RU− vs. aged RU+); one-way ANOVA/Tukey’s multiple comparisons test.
(e) Immunostaining of nuclei isolated via isotropic fractionation from brains of young (10-day-old) and aged (30-day-old) elavGS>UAS-BNIP3 flies with or without RU486-mediated transgene induction from day 5 onward, showing ELAV (green channel) and DNA (blue channel, stained with To-Pro-3). Scale bar is 50 μm.
(f) Quantification of neuronal (elav+) nuclei isolated from brains via isotropic fractionation as shown in (e). n = 6 biological replicates per condition with 3 pooled brains per replicate. *p=0.0263, **p=0.0098; one-way ANOVA/Tukey’s multiple comparisons test. Data are presented as scatter plots overlaying mean values +/− SEM.