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. 2021 Feb 1;4(3):e202000693. doi: 10.26508/lsa.202000693

Figure 3. Increasing miR-8 levels in gliomas impairs ImpL2 overexpression and synapse loss.

Figure 3.

(A, B) Confocal microscopy images of adult brain showing the expression pattern of miR-8 in repo>UAS-LacZ control animals compared with repo>UAS-dEGFRλ, UAS-dp110CAAX flies using miR-8 sensor (see the Materials and Methods section). (A′, B′) Glial membrane is marked in red. (C) Quantification and statistical analysis of miR-8 expression based on the GFP pixel intensity in at least N = 3 per genotype (ANOVA, post hoc Bonferroni) (scale bar, 50 μm). GFP control levels were normalized to one and GFP increase in glioma brains was transformed into negative values to indicate the relation between the increase in GFP and the reduction in miR-8 levels of expression. (D, E, F) Confocal microscopy images of 7-d-old adult brains from the following genotypes: (D) repo>UAS-LacZ (Control), (E) repo>UAS-dEGFRλ, UAS-dp110CAAX (Glioma), (F) repo>UAS-dEGFRλ, UAS-dp110CAAX, UAS-miR-8 (Glioma miR-8) animals, in all cases combined with ImpL2-MIMIC GFP transgene (scale bar, 50 μm). (D′, E′, F′) Merge with glial membrane in red and DAPI staining. (G) Quantification and statistical analysis of co-localization rate between ImpL2-MIMIC GFP and glial membrane in at least N = 10 per genotype (ANOVA, post hoc Bonferroni). (H, I, J, K) Adult neuromuscular junction (NMJ) from (H) repo>UAS-LacZ (controI), (I) repo>UAS-dEGFRλ, UAS-dp110CAAX (Glioma), (J) repo>UAS-dEGFRλ, UAS-dp110CAAX, UAS-miR-8 (Glioma miR-8), and (K) repo>miR-8 (Glia miR-8), active zones marked in green. (L) Quantification and statistical analysis of active zones in at least N = 10 per genotype (ANOVA, post hoc Bonferroni) (scale bar, 50 μm). (M, N, O, P) Confocal microscopy images of adult brains from 7-d-old flies of (M) repo>UAS-LacZ, (N) repo>UAS-dEGFRλ, UAS-dp110CAAX, (O) repo>UAS-dEGFRλ, UAS-dp110CAAX, UAS-miR-8 and (P) repo>UAS-miR-8 genotypes, glial nuclei marked in green. (M′, N′, O′, P′) Glial membrane is shown in red. (Q, R) Quantification of (Q) glial cells and (R) glial membrane volume for at least N = 9 per genotype (ANOVA, post hoc Bonferroni) (scale bar, 100 μm) (*P-value < 0.05**P-value < 0.01, ***P-value < 0.001).