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. 2023 Nov 13;11:1297910. doi: 10.3389/fcell.2023.1297910

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Copyright © 2023 Katkat, Demirci, Heger, Karagulle, Papatheodorou, Brazma and Ozhan.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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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+/−,HRAS ^G12V :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+/−,HRAS ^G12V :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+/−,HRAS ^G12V :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.