Figure 2

Figure 2. Induction of tumor metastasis in twist1a+/xmrk+ transgenic zebrafish via treatment with Dox in high concentrations. Twist1a+, xmrk+, and twist1a+/xmrk+ transgenic zebrafish as well as wild-type control were treated with 60 μg/mL Dox and 1 μg/mL 4-OHT starting from 3 mpf. Samples were collected at 2 and 4 wpi. (A) Representative images of transgenic zebrafish at 4 wpi. The left column displays external appearance, the middle column shows internal abdominal organs with the livers outlined, and the right column depicts H&E staining of liver sections. Scale bar: 50 or 200 μm. Compared with the wild-type group, (B) the body lengths of transgenic zebrafish differed significantly at 2 wpi but not at 4 wpi, whereas (C) the body weights of transgenic zebrafish differed significantly at 2 and 4 wpi. (D) Kaplan–Meier survival curves of days post-induction plotted against percent survival to 4 wpi. (E) Immunofluorescence analysis of liver tumor metastasis in twist1a+/xmrk+ transgenic zebrafish at 4 wpi. (F) Histological examination confirmed that xmrk+ and twist1a+/xmrk+ transgenic zebrafish developed HCC or metastatic HCC at 2 and 4 wpi, whereas normal liver histology was observed in all twist1a+ and wild-type siblings. Differences among variables were assessed using Student’s t-tests or one-way ANOVA. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001.