Nis elements f 3

Liver tissue sections were made from paraffin-embedded tissues. The sections were stained with hematoxylin and eosin, and the histopathologic changes were observed under a light microscopy (Nikon ECLIPSE 50i) and the micrographs were taken with a digital camera (Nikon Digital Sight DS-Fi1, Nikon Corporation, Minato-ku, Tokyo, Japan) and NIS Elements F 3.0 (Nikon Corporation, Minato-ku, Tokyo, Japan) image acquisition software. The hepatocytes damages were conducted using a score system described by Kleiner et al [50 (link)]. Briefly, the damage score consisted of scores for glycogenated nuclei (graded 0-1, from none or rare to many continuous patches), liver cells ballooning injury (graded 0-2, from absent to severe ballooning injury) and inflammatory cells filtrate (graded 0-3, from absent to transmural). Three tissue sections from each animal were coded and examined by two blinded observers to prevent observer bias.
Biochemical parameters such as Total protein, Albumin, Aspartate aminotransferase, Alanine aminotransferase and Lactate dehydrogenase were determined using a biochemical analyzer (SPOTCHEM™EZ SP-4430, Arkray, Kyoto, Japan).

The morphology of the zebrafish larvae (otolith and body curvature) was imaged at 4 dpf using a Nikon AZ100 microscope (Nikon, Tokyo, Japan). The larvae were raised in Petri dishes and transferred to a new dish at 5 dpf for behavioral analysis. After allowing adaptation to the new environment for 5 min, locomotion was video-recorded for 3 min using a Nikon DS-Fil1 digital camera and processed with NIS-Elements F3.0 (Nikon).

Whole-mount in situ hybridization (WISH) was performed using probes for cdkn1a, mdm2, aaas, eftud2, gss, huwe1, mettl16, ice1, prpf3, prpf4, snrnp200, prpf6, snapc4, prpf8, snrpe, prpf31, sf3b4, eif4a3, tp53, pcna, her4.1 (Supplementary Table 3)^{60 (link)}. Staged embryos were fixed overnight in 4% PFA, and then dehydrated in a methanol gradient. Embryos were then rehydrated in phosphate-buffered saline containing 0.1% Tween-20 (PBST). Embryos were permeabilized by proteinase K digestion and then hybridized with digoxin-labeled probes overnight at 70 °C. The next day, embryos were washed in a preheated mixture of 50% saline sodium citrate containing 0.1% Tween-20 and 50% hybridization solution at 70 °C. Embryos were washed again at room temperature and incubated in staining solution in the dark until sufficient staining appeared. Embryos were mounted in glycerol and were visualized using a Nikon AZ100 microscope (Nikon, Tokyo, Japan). Images were captured using a Nikon DIGITAL SIGHT DS-Fil1 digital camera (Nikon) and processed with NIS-Elements F 3.0 (Nikon).