Figure 1. Novel TIE:EGFP zebrafish enhancer reporter is expressed in advanced melanomas.
(A) TGFb-induced enhancer (TIE) used to construct TIE:EGFP reporter determined by H3K27ac, SMAD2/3, and MITF ChIP-seq peaks in A375s+/-TGFB1. There is unique H3K27ac and SMAD2/3 binding upon stimulus. (B) TIE:EGFP expression throughout zebrafish development. Scale bars represent 500 µm. (C) TIE:EGFP expression across melanomagenesis indicated by arrowheads. Representative images shown. Additional tumors shown in Figure 1—figure supplement 3. Illustrated fish diagram in (C) created with BioRender.com, and published using a CC BY-NC-ND license with permission.
© 2024, BioRender Inc
Figure 1 was created using BioRender, and is published under a CC BY-NC-ND license. Further reproductions must adhere to the terms of this license.
Figure 1—figure supplement 1. TIE:EGFP zebrafish enhancer reporter is inducible and specific.
(A) TIE:EGFP reporter is induced upon electroporation with ubi:caSMAD2 and ubi:caSMAD3 in adult zebrafish flank skin. Ubi:BFP was used as a control for varying electroporation efficiency. TIE:EGFP intensity/BFP area was quantified (right). Two-tailed unpaired Welch’s t-test was used to calculate significance. Representative images shown. n=8 fish per condition. (B) TIE:EGFP embryos treated at 1 day post fertilization for 24 hr with E3 (zebrafish water), DMSO (vehicle control), 50 µM or 100 µM TGFb inhibitor SB431542. TIE:EGFP channel (top) and white light (bottom). Representative images shown. Scale bars represent 500 µm. (C) In situ hybridization (ISH) was performed probing for GFP in embryos treated with DMSO (n=26), 50 µM (n=30), or 100 µM (n=31) TGFb inhibitor SB431542 for 24 hr starting at 1 day post fertilization. Embryos were grouped into three qualitative groups based on GFP intensity: low, medium, and high, and the percentage of embryos in each are graphed below. (D) Representative image of mitfa:mCherry+ zebrafish fin melanocytes expressing TIE:EGFP. The uppermost EGFP+ region not overlapping with mCherry signal is residual spinal expression of TIE:EGFP. (E) Early melanomas, indicated by arrowheads, do not express TIE:EGFP. The EGFP+ region not overlapping with mCherry signal is residual brain expression of TIE:EGFP. Representative images shown. Illustrated fish diagrams in (A, D, E) created with BioRender.com, and published using a CC BY-NC-ND license with permission.
© 2024, BioRender Inc
Figure 1—figure supplement 1 was created using BioRender, and is published under a CC BY-NC-ND 4.0. Further reproductions must adhere to the terms of this license.
Figure 1—figure supplement 2. Treatment with human recombinant TGFB1 activates the TGFb pathway in human melanoma cells.
(A) (Left) RNA-sequencing indicated that following a 2-hr treatment with 10 ng/mL TGFB1, 223 genes were significantly up-regulated (q<0.05), including typical TGFb target genes such as SMAD7, JUNB, and PMEPA1, while 94 genes were down-regulated. This Volcano plot indicates top up- and down-regulated genes upon TGFB1 treatment. (Right) Hallmark gene set enrichment analysis (GSEA) of genes ranked by log2 fold-change (log2fc) confirmed that TGFb was the top up-regulated pathway (q=0). RNA-seq was performed in triplicate. (B) TIE:EGFP+ melanoma #2 expressing MCR:MCS. TIE:EGFP expression across melanomagenesis. Representative images shown. Illustrated fish diagrams in (B) created with BioRender.com, and published using a CC BY-NC-ND license with permission.
© 2024, BioRender Inc
Figure 1—figure supplement 2 was created using BioRender, and is published under a CC BY-NC-ND 4.0. Further reproductions must adhere to the terms of this license.
Figure 1—figure supplement 3. TIE:EGFP expression across melanomagenesis in several additional TIE:EGFP;Tg(mitfa:BRAFV600E);p53-/-;mitfa-/-;MCR:MCS;mitfa:mCherry;tyr-/- fish.
Clusters of TIE:EGFP+ cells indicated by white arrowheads.