Fig. 7

Tnfa inhibits the autophagy in zebrafish larvae.

(A) Workflow representing the experimental design. B) Zebrafish GFP-LC3 transgenic embryos were injected with Tnfa or Std mos at the one-cell-stage of development. After 48 hours, a group of larvae injected with Std-mo was immersed in a bath with 1 μM Rapa and was freshly added every 24 h. The remaining larvae were divided in two and challenged by bath immersion with 10⁹ TCID₅₀/ml SVCV SVCV or RPMI alone. After 72 hours of infection (5 dpf), larvae were collected, anesthetized with 0.16 mg/ml tricaine, mounted in 1% low melting point agarose supplemented with 0.16 mg/ml tricaine and images of the whole larvae taken using a Leica MZ16F fluorescence stereo microscope. Numbers in pictures represent the animals with the shown phenotype per total analyzed animals. (C) Zebrafish larvae were injected with morpholino (mo) Tnfa (Tnfa-MO) or Std (Std-mo) 1 hour post fertilization (hpf). After 36 hours, a group of larvae injected with Std-mo was immersed in a bath with Rapamycin and the remaining larvae (72 each group) were then divided in two and challenged by bath immersion with SVCV as above. After 48 hours of infection samples were recollected and LC3-I and LC3-II were detected by western-blot using an anti-LC3 antibody and the densitometry values were used to calculate LC3-II/ LC3-I ratios, represented as black bar graphs. Actin bands were detected as a protein load internal control using an anti-actin antibody. Data are shown as the mean±S.E.M. of 3 independent experiments. p<0.05.