DLD role in metabolic pathways, generation of the dldh–/– zebrafish model, and confirmation of Dldh protein deficiency in dldh–/– zebrafish larvae. (A) Scheme representing enzyme complexes incorporating a functional DLD (E3) subunit: PDHc is the gateway enzyme to the TCA cycle, and AKADHc and BCKADHc are essential enzymes in lysine and BCAA catabolism, respectively. (B) CRISPR/Cas9 construction of dldh–/– zebrafish. Schematic showing where sgRNA targets exon 1 after start codon (ATG); the sgRNA was injected 1 hpf. (C) Sanger confirmation of 5 bp deletion in dldhcri3 zebrafish. (D) Immunoblot of Dldh protein level in 7 dpf WT, dldh+/–, and dldh–/– larvae. (E) ImageJ quantification of Dldh, CS, VDAC, and tubulin protein levels in WT vs. 7 dpf dldh–/– larvae, showing significantly decreased Dldh expression in dldh–/– larvae and increased VDAC levels in dldh+/– and dldh–/–; n = 3; *P < 0.05 and **P < 0.01, unpaired Student’s t test with the Bonferroni correction. Data are shown as mean ± SD.
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