Generation of in vivo knockdown models of CBS and CGL, regulators of transsulfuration pathway involved in Hcy catabolism. (A) Simplified diagram showing the enzymes and metabolites of transsulfuration pathway (B) Representative western blot of 2 dpf embryos lysates showing that compared with SC MO injected embryos, CBS protein level is downregulated in CBS MO injected embryos. Expression of CGL, the other enzyme of the same pathway, remained unaltered. As a loading control β-actin was used. Corresponding bar plot showing fold change in protein expression (normalized to β-actin) as determined by densitometric analysis of the protein band. (C) Bar plot revealing no statistically significant difference in the viability of scrambled control and Hyperhomocysteinemic CBS MO embryos. (D) Bright field images exhibiting no apparent gross morphological defect in CBS morphants compared with scrambled Mo injected embryos of 2 dpf. Scale bar, 0.25 mm. (E) Representative western blot of embryo lysates at 2 dpf showing reduced protein level of CGL in the embryos injected with CGL MO. Expression of CBS, the upstream protein of the same pathway, remained unaltered. β-actin was used as a loading control. Corresponding bar diagram showing densitometric analysis of the fold change in protein expression (normalized to β-actin). (F) In comparison to SC MO injected embryos, viability of CGL MO injected embryos were not altered significantly. (G) Representative bright field images showing absence of any gross morphological defect in CGL MO injected embryos compared with SC MO injected ones at 2 dpf. Scale bar, 0.25 mm. Data are shown as Mean ± SEM with n ≥ 3. ** p ≤ 0.01, **** p ≤ 0.0001 and ns is non-significant (p > 0.05).
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