Fig 1: Caspase-3 facilitates melanoma metastasis in vivo.A Workflow for the generation of primary zebrafish melanomas upon microinjection of the transposon-based MiniCoopR vector into one-cell stage embryos. B Western blot analysis showing the expression levels of Caspase-3 (CASP3) and Green Fluorescent Protein (GFP/Ctl) in primary zebrafish melanoma tumors. C Tumor-free survival curves of Tg (mitfa:BRAFV600E), tp53−/−, mitfa−/− zebrafish injected with vectors for the overexpression of CASP3 or GFP (Ctl). Statistical analysis was performed using the log-rank test. D Workflow for the transplantation of primary zebrafish melanomas into the dorsal cavity of secondary recipient Casper fish. E Representative images of Casper fish transplanted with primary zebrafish melanomas overexpressing CASP3 or GFP. Arrows indicate the location of disseminated melanoma cells. F Quantification of the percentage of secondary recipients with tumor spreading, comparing control GFP (n = 6) and CASP3 (n = 7) overexpressing tumors. Data are presented as mean ± standard deviation (S.D.); statistical analysis was performed using two-tailed t-test.
Fig 2: Caspase-3 controls melanoma cell motility.A Quantification of cell adhesion potential of parental and CASP3-depleted WM793 cells (n = 3, one representative experiment shown, two-way ANOVA statistical test; Statistical significance: ns - P > 0.05; * - P ≤ 0.05; ** - P ≤ 0.01; *** - P ≤ 0.001; **** - P ≤ 0.0001). B Time-lapse cellular tomography in WM793 cells. C Schematic representation of the wound healing assay using the IncuCyte live cell imager, which assesses the migratory ability of melanoma cells to close a wound. Quantification of migration potential in parental and CASP3-knockdown WM793 (D) and WM852 (E) cells through kinetic wound area measurement (n = 3, one representative experiment shown, two-way ANOVA statistical test; Statistical significance: ns - P > 0.05; * - P ≤ 0.05; ** - P ≤ 0.01; *** - P ≤ 0.001; **** - P ≤ 0.0001). F Schematic representation of the invasion assay through matrigel. Measurement of the invasion potential of WM793 (G) and WM852 (H) depleted or not for CASP3 (n = 3, one representative experiment shown, two-way ANOVA statistical test; Statistical significance: ns - P > 0.05; * - P ≤ 0.05; ** - P ≤ 0.01; *** - P ≤ 0.001; **** - P ≤ 0.0001). I Schematic representation of the chemotaxis assay. Measurement of control and CASP3-knockdown WM793 (J) and WM852 cells (K) chemotaxis potential (n = 3, one representative experiment shown, two-way ANOVA statistical test; Statistical significance: ns - P > 0.05; * - P ≤ 0.05; ** - P ≤ 0.01; *** - P ≤ 0.001; **** - P ≤ 0.0001). L The zebrafish model of cancer metastasis. M DiD-labelled CASP3 knockdown and DiO-labelled control WM793 cells were pre-mixed in equal numbers and injected in the zebrafish embryos yolk sac as shown in (L). A representative epifluorescence image of a whole embryo shows perivitelline homing and caudal blood vessels invasion of cancer cells. White arrows point towards invading cells. N Quantification of invaded metastatic cells per zebrafish embryo, (data represent mean with SEM, n = 39 embryos from three independent experiments, t-test, ***p < 0.001).
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