Effects of PH on the kras^G12V-induced development of HCC in zebrafish. (A) Schedule of surgeries and Dox treatment in the experiment. Right after PH or sham surgery, kras+ male zebrafish were exposed to 20 mg/L Dox to induce the overexpression of oncogene kras^G12V in the liver. (B) Representative photos of PH/sham surgery-treated kras+ zebrafish 3 days and 7 days after Dox induction. (C) Measurement and comparison of the 2D area of the kras+ livers in mm² based on (B) after being normalized with the body length in cm (n ≥ 4). Circles, squares, and triangles indicate values of individual samples. (D) Representative H&E staining images of the PH/sham surgery-treated kras+ zebrafish livers 3 days after Dox induction (n = 6). Examples of morphology associated with HCC and hyperplasia are indicated with symbols. Red arrowheads: irregular nuclei with prominent nucleoli; white arrowheads: nuclei with multiple nucleoli; black arrowheads: enlarged nuclei with prominent nucleoli; red bracket: two-cell hepatocyte plate structures. (E) Quantification of HCC histology observed in kras+ zebrafish from each treatment group on Day 3 based on (D). (F) Representative H&E staining images of the PH/sham surgery-treated kras+ zebrafish livers 7 days after Dox induction (n = 6). Scale Bar = (B) 1 mm and (D,F) 20 μm. ns p > 0.05, * p ≤ 0.05.

Effects of PH on the progress of HCC-associated characteristics in kras+ zebrafish. (A) IF staining of PCNA and HNF4α in the PH/sham surgery-treated kras+ zebrafish liver 3 days after Dox induction. (B) Quantification of PCNA and HNF4α-double positive proliferating hepatocytes based on (A) (n = 4). Circles indicate values of individual samples. (C–E) Expression of (C) ccnb1 and cdk1, (D) col1a1b, lama5, (E) acta2 and tgfb1b in the PH/sham surgery-treated kras+ zebrafish livers 3 days after Dox induction as determined by RT-qPCR (n = 4). (F) Expression of kras^G12V in the PH/sham surgery-treated kras+ zebrafish livers 3 days after Dox induction as determined by RT-qPCR (n = 3). Scale Bar = 20 μm. ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Effects of PH on oxidative stress and neutrophil activity in the kras+ zebrafish liver during the kras^G12V-induced development of HCC. (A) IHC staining of HIF1α in PH/sham surgery-treated kras+ livers on Day 5. (B) Quantification of HIF1α-positive liver tissue based on (A) (n = 5). The HIFα positive fraction was determined by measuring the DAB-stained area versus the total tissue area with imageJ plugin color deconvolution. (C) Expression of hif1aa in PH/sham surgery-treated kras+ livers on Day 3 as determined by RT-qPCR (n = 5). (D) Expression of hif1aa in WT male livers within 72 h after PH/sham surgery as determined by RT-qPCR (n = 3). (E) Fluorescence images of PH/sham surgery-treated kras/lyz+ livers following Dox induction. (F) Quantification of DsRed+ neutrophils in the liver based on (E) (n ≥ 4). (G) Expression of cybb in PH/sham surgery-treated kras/lyz+ livers on Day 3 as determined by RT-qPCR (n = 4). Scale Bar = 20 μm. Circles, squares, and triangles in (B,F) indicate values of individual samples. ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01.

Comparison of DEGs during kras^G12V-induced HCC development and PH-induced liver regeneration in zebrafish. (A) Workflow of the differential expression analysis based on the RNA-Seq reads. (B) Experimental design for comparing the transcriptomic changes in the kras+ livers with those in the PH livers with the quantification of total DEGs. (C,D) Venn diagrams showing the overlaps of (C) upregulated genes and (D) downregulated genes between the kras+ male liver on Day 5 and the PH male liver on Day 1. (E,F) Heatmaps of genes that were significantly (A) upregulated and (B) downregulated in both the kras+ liver on Day 5 and the PH liver on Day 1 based RNA-seq data. (G,H) Expression of (G) fgf13b and (H) s100a1 in the kras+ male livers 24 h after PH/sham surgery as determined by RT-qPCR (n = 4). ns p > 0.05, ** p ≤ 0.01.

Comparison of changes in biological functions and pathways between the kras+ zebrafish liver and the PH liver. (A) Comparison between the changes in the activity of Hallmark gene sets in the kras+ liver on Day 5 following Dox induction and those in the PH livers on Day 1 following PH based on the GESA of Hallmark gene sets. The color key indicates the direction of changes (red: upregulation; green: downregulation) and significance (adjusted p-value). (B,C) GESA of KEGG pathway in (B) kras+ livers on Day 5 following Dox induction and (C) PH livers on Day 1 following PH. The size of the dots indicates the number of leading-edge genes in the corresponding gene set. The color key indicates significance in adjusted p-value. Gene Ratio is the ratio of leading-edge genes versus all genes in the gene set.

Comparison of changes in biological functions and pathways between the kras+ zebrafish liver and the PH liver. (A) Comparison between the changes in the activity of Hallmark gene sets in the kras+ liver on Day 5 following Dox induction and those in the PH livers on Day 1 following PH based on the GESA of Hallmark gene sets. The color key indicates the direction of changes (red: upregulation; green: downregulation) and significance (adjusted p-value). (B,C) GESA of KEGG pathway in (B) kras+ livers on Day 5 following Dox induction and (C) PH livers on Day 1 following PH. The size of the dots indicates the number of leading-edge genes in the corresponding gene set. The color key indicates significance in adjusted p-value. Gene Ratio is the ratio of leading-edge genes versus all genes in the gene set.

Regulation of ribosome protein genes and ribosomal RNAs during kras^G12V-induced zebrafish HCC development and PH-induced zebrafish liver regeneration. (A) Regulation of genes under the KEGG ribosome pathway in kras+ livers after 5 days of Dox induction. (B) Regulation of genes under the KEGG ribosome pathway in PH livers at 1 day post-PH.

The effect of s100a1 knockdown on the development of kras^G12V-induced HCC in zebrafish larvae. (A) Experiment design of s100a1 knockdown and oncogene induction in Lipan larvae and kras+ larvae. (B) Western blot of S100A1with the total protein extracted from MO-injected zebrafish embryos at 24 hpf. β-actin served as a loading control. Each protein sample was pooled from 15 zebrafish embryos. The quantification of the intensity of S100A1normalized by the intensity of β-actin was at the lower panel. The uncropped image of blots is shown in Figure S1. (C) Fluorescence images of morpholino-treated Lipan and kras+ larvae after 48 h of Dox treatment. (D) Measurement of liver size based on the fluorescence in (B) (n ≥ 8). Circles, squares, and triangles indicate values of individual samples (E) Expression of sox5, sox9a, and sox9b in the kras+ livers at 24 h after PH/sham surgery as determined by RT-qPCR (n = 3). Scale Bar = 200 μm. ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01.