Scamp5a knockout resulted in apoptosis and dopamine neurons loss in zebrafish brain. A, B 72 hpf WT (n = 16) and scamp5a−/− (n = 24) larvae were stained with acridine orange (AO) to label apoptosis cells in vivo. A Representative images of AO staining in the larvae brains as indicated. Scale bars: 100 μm. Enlarged images of white boxed areas are shown in lower left corner. B Relative apoptosis signal level of AO staining in the brain areas of the WT and scamp5a−/− larvae. Scamp5a KO resulted in apoptosis in zebrafish brain. C, D Representative images (C) and Representative images (C) and relative analysis (D) of the th1-positive dopamine neurons level in 72 hpf larvae. WT (n = 14), scamp5a−/− larvae (n = 18) and scamp5a−/− larvae injected with human GFP-SCAMP5 (n = 27). Dopamine neurons are indicated by whole-mount in situ hybridization (WISH) staining of th1 mRNA. Scale bars: 100 μm. th1, marker gene for dopamine neuron. Human SCAMP5-WT could rescue the reduced dopamine neuron in scamp5a−/− larvae. E RT-qPCR analysis of the transcript level of th1 in 5 dpf larvae brains. The relative transcript level of th1 in scamp5a−/− larvae was compared to that in WT. Housekeeping gene actb was used as internal control for RT-qPCR assay. n = 30 larvae per group for mRNA extracts. F RT-qPCR analysis of apoptosis-related genes in 5 dpf larvae brains. The relative transcript levels of genes in scamp5a−/− larvae were normalized to those in WT. n = 30 larvae per group for mRNA extracts. G, H WT, scamp5a−/− larvae and scamp5a−/− larvae injected with plasmid as indicated at the one-cell stage. Brains extracts from 5 dpf larvae as indicated were harvested for Western blot analysis. G Lysates were probed with indicated antibodies. The primary antibody was anti-Bcl2, anti-Bad, anti-caspase3, and anti-cleaved-caspase3. n = 20 larvae per group for proteins extracts. Plasmid: GFP tag or human GFP-SCAMP5. H Quantification of relative expression levels of apoptosis-related factors compared to those in WT. Tubulin5 were used as a loading control. I, J Western blot analysis of β-synuclein in 5 dpf WT and scamp5a−/− larvae brains. I Lysates were probed with indicated antibodies. The primary antibody was anti-β-synuclein. Brains extracts from 5 dpf larvae as indicated were harvested for Western blot analysis. n = 20 larvae per group for proteins extracts. J Relative expression of β-synuclein in scamp5a−/− larvae compared to that in WT larvae. GAPDH was used as a loading control. The data in panel B, D–F, H and J are presented as mean ± SEM. n ≥ 3 independent experiments. Significance levels are denoted as follows: *P < 0.05, **P < 0.01, ***P < 0.001
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