|
Fig. 5 Metastasis repression by PA5 in zebrafish melanoma models. (a) Metastatic cells were labeled with a viable dye and were injected in the zebrafish yolk at 2 dpf. One day after cell injection, zebrafish displaying cells in the circular vessels were discarded. Three days after cell injection, a solution of PA5 (500 nM or 1,000 nM) was injected into the zebrafish yolk. Zebrafish were monitored until 4 dpt (7 dpi). Metastases were classified as follows: (i) in place, cells confined to the site of injection; (ii) initial metastases, cells spread from the yolk to near organs (heart, swim bladder, and pharynx); and (iii) full metastases, cells in distant organs, such as brain, skeletal muscle, and trunk. (b) M121224- and M130425-labeled cells (red) are shown at the time of PA5 injection. Bar = 1 mm (c) Zebrafish were monitored each day up to 4 dpt (7 dpf) to evaluate the mortality rate after treatment. The number of fish on 0 dpt were set as 100% of zebrafish viability. (d, e) Localization of the stained cells (red, indicated with white arrows) was evaluated, and the metastasis was quantified. Bar = 1 mm. (f, g) Cell fluorescence intensities were measured by ImageJ software and normalized on cell fluorescence at time 0. Data represent the means ± SD of three independent biological experiments. Student’s t-test was used for statistical analyses. ∗∗P < 0.01; ∗0.01 < P < 0.05. CTRL, control; dpf, days post fertilization; dpi, days post-injection; dpt, days post-treatment; PA, protein kinase G activator.