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. 2021 May 16;18(2):309–319. doi: 10.1080/15548627.2021.1924038

Figure 5.

Figure 5.

AHCYL1 is required for the autophagy inhibition by SAH in zebrafish. (A) A flowchart for the generation of ahcyl1-KO zebrafish using the CRISPR/Cas9 system to analyze autophagy with or without SAH. (B) An example of 26 h zebrafish embryos expressing RFP-LC3. The fertilized eggs from the cross of wild-type and chimeras were injected with RFP-LC3 mRNA and 0/5 mM SAH at one cell stage. Twenty-six h later, the embryos expressing RFP-LC3 were selected and fixed for analysis. (C) An example of ahcyl1-KO heterozygotes. A deletion of 71 bp. The fixed embryos were dissected into tails and heads. The tails were used for DNA extraction and sequencing. The heads were analyzed for the RFP-LC3. (D) and (E) ahcyl1 is required for the decrease of LC3 puncta by SAH. RFP-LC3 puncta in the brain areas of embryos were analyzed. Injection of SAH decreased LC3 puncta in ahcyl1+/+ zebrafish and the decrease was less in ahcyl1± zebrafish. Bars and error bars represent mean values and SDs, respectively. The Two-way ANOVA was used for E. ns represents no significance, defined as P > 0.05. N = 10 in E.