Activation of canonical Wnt or TGF-β/BMP signaling pathways suppresses migration and proliferation of human melanoma cells in vivo(A) Representative fluorescence microscope images of 7 dpf zebrafish larvae xenografted with human melanoma cells (SK-MEL-28) labeled with DiO (pseudo-color green) at 2 dpf and treated with DMSO, BIO or ISL. (B) Dot plot showing the number of invading cells at 7 dpf larval xenografts with micrometastasis. Each dot represents one larval xenograft (DMSO n = 44, BIO n = 38, ISL n = 41). (C) Representative confocal microscope images of anti-phospho-histone H3 (green) staining of 7 dpf zebrafish larvae xenografted with SK-MEL-28 cells (red) at 2 dpf and treated with DMSO, BIO, or ISL. (D) Dot plot showing the percentage of mitotic figures in each treatment. Each dot represents mitotic figures counted in each z-stack slice divided by the number of DiO + DAPI + nuclei in (C). Larvae were counterstained for DAPI. Scale bars are 50 μm. Statistical significance in B and D was evaluated using a one-way ANOVA test ****p < 0.0001. (E) Western blot of 5 dpf Tg(mitfa+/−,HRASG12V:GFP) zebrafish larvae treated with BIO or ISL for total and phospho-ERK (p-ERK). Tg (mitfa+/−) larvae were used as control. Graph shows the average relative density ratios of p-ERK to total ERK from three independent experiments. (F) qPCR on Tg(mitfa+/−,HRASG12V:GFP) zebrafish larvae treated with BIO or ISL showed reduced pigmentation marker genes tyr and dct expression at 5 dpf. Tg (mitfa+/−) larvae were used as control. rpl13 was used as the housekeeping control gene. (G) Melanin content of Tg(mitfa+/−,HRASG12V:GFP) zebrafish larvae treated with BIO or ISL. Error bars represent ±standard error of the mean (SEM, n = 6). Statistical significance was evaluated using an unpaired t-test. *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001. DMSO, dimethyl sulfoxide; ISL, isoliquiritigenin; dpi, days post-injection; tyr, tyrosinase; dct, dopachrome tautomerase.
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