(a) Protocol used to induce TO(krasG12V)T/+ expression in the livers of developing zebrafish larvae. (b) RT-quantitative PCR (RT-qPCR) analysis of ahctf1 mRNA levels in pooled micro-dissected larval livers (n = 3 biological replicates). (c) Representative three-dimensional reconstructions of 2-CLiP and dox-treated TO(krasG12V)T/+ larval livers of the indicated ahctf1 genotype. Scale bar 25 µm. (d) Impact of ahctf1 heterozygosity on liver volume in 2-CLiP and TO(krasG12V)T/+ larvae (n ≥ 20). (e) Impact of ahctf1 heterozygosity on liver-to-body mass ratio of adult TO(krasG12V)T/+ zebrafish in the presence or absence of dox treatment (n = 10). (f) Histological sections of adult male TO(krasG12V)T/+ zebrafish livers of the indicated ahctf1 genotype and dox treatment, stained with haematoxylin and eosin. In vehicle-treated adults, hepatocytes are densely packed and well differentiated. White arrows point to sections through blood vessels containing red blood cells. Meanwhile, the hepatocytes in dox-treated animals are poorly differentiated and exhibit multiple cytological abnormalities, including pyknotic nuclei (arrowheads) and vacuolation (black arrows). Scale bar 25 µm. (g) Western blot of Ras and Gapdh protein signals in total input lysates (50 μg) of TO(krasG12V)T/+ larvae of the indicated ahctf1 genotype and dox treatment. (h) Western blot of active Ras-GTP protein signals in lysates following active Ras pull-down.
