Skip to main content
. 2021 Jul 24;24(8):102899. doi: 10.1016/j.isci.2021.102899

Figure 4.

Figure 4

Increased autophagy, apoptosis, and secondary necrosis in Pss mutants

(A) Autophagy in the brain detected by acridine orange (AO) (excitation/emission: 561/579–620). (a, a') 1-day-old WT. (b, b') 1-day-old Pss32/15. (a, b) Brain images are shown in 'fire' mode for better contrast. (c) Boxplots of quantified fluorescence intensities. At least five specimens were used for the quantification. Scale bar, 100 μm. ∗p < 0.05.

(B) Dying cells detected by AO in the brains. AO binding with ssDNA emits fluorescence at a longer wavelength than 650 nm (excitation: 488 nm). (a, a') 5-day-old WT. (b, b') 5-day-old Pss32/15. (a, b) Brain images are shown in 'fire' mode for better contrast. (c) Boxplots of quantified fluorescence intensities. At least five specimens were used for the quantification. Scale bar, 100 μm. ∗p < 0.05.

(C) TEM images of ommatidia of (a) 5-day-old WT and (b) Pss32/15 flies. The inter-ommatidial area that is composed of pigment cells is greatly expanded in Pss32/15 flies. Red boxes in a, b are magnified in a', b'. R: rhabdomeres, m: mitochondria. Autophagic bodies are indicated by asterisks (∗). (c) A bar graph showing numbers of autophagosomes/autolysosomes per ommatidium. Pss32/15 flies display about 4.2 times more autophagosomes/autolysosomes per ommatidium than WT flies. Twenty specimens were used for the quantification. The error bars represent mean ± SD (∗∗∗p < 0.001). The numbers above the error bars indicate mean values. Scale bar, 5 μm.

(D) Detection of apoptotic cells with anti-cleaved caspase 3 antibody. (a, d) WT. (b, e) Pss32/+. (c) Boxplots of quantified immunofluorescent puncta per 104μm2 of 1-day-old flies' central brains ((total puncta number/area)x104). (f) Boxplots of quantified immunosignal area in cortex of 5-day-old flies' brains ((signal positive area/total cortex area)x100). AL: antennal lobe, CB: central brain, CT: cortex, OL: optic lobe. Scale bar, 50 μm.

(E) Secondary necrosis in Pss mutants. WT and Pss32/15 brains were stained with a necrosis detector. (a) While only faint signals appeared in the WT brains, (b) strong and widespread signals appeared in the mutant brains. (c) Boxplots of quantified fluorescence intensities. At least four specimens were used for the quantification. Scale bar, 100 μm. ∗p < 0.05.