In vitro and in vivo characteristics of zebrafish mthfd1L. (A) The complete recombinant zebrafish Mthfd1L, encompassing the N-terminal signal peptide, was co-localized with mitochondria. Huh-7 cells transfected with zmthfd1L/pcDNA 3.1 myc-His (zMthfd1L) and pcDNA 3.1 myc-His (Mock) (left panel) and primary cultured zebrafish embryonic cells from embryos injected with mthfd1L cRNA (right panel) were stained with DAPI for nucleus, Mitotracker Deep Red 633 for mitochondria, and anti-His for zebrafish Mthfd1L. (B) The cytosolic and mitochondrial fractions of 293 cells transfected with zmthfd1L/pcDNA 3.1 myc-His and Mock analyzed with Western blotting confirmed the mitochondrial localization of zebrafish Mthfd1L. (C) The neural tube defect (NTD)-like phenotype was found in mthfd1L morphant (MO), but not in wild-type larvae (Ctl) at 3 dpf. (D,E) The eye size of mthfd1L morphants with (MO+cRNA)/without (MO) co-injecting synthesized mthfd1L capped-mRNA (cRNA) for rescue were examined and quantified at 3 dpf. Embryos were injected with translational MO (tMO) or splicing site MO (sMO) at 1–4-cell stage. (F) 1 dpf zebrafish embryos injected with mthfd1L cRNA were collected for embryonic lysates and subjected to Western blotting analysis. (G–I) Mthfd1L morphants at 3 dpf were analyzed with HPLC for folate composition. c, cytosolic fraction; m, mitochondrial fraction; α-tub, cytosolic marker; and Cox4, mitochondrial marker. MO, wild-type embryos injected with 10 ng mthfd1L MO; MO+cRNA, wild-type embryos co-injected with 10 ng mthfd1L MO and 1 ng mthfd1L synthesized RNA simultaneously. ***p < 0.001; **p < 0.01; *p < 0.05.
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