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Fig. 3
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Fig. 3. Combined MYCN-RRM2 overexpression in zebrafish sympathetic neuroprogenitor cells results in accelerated neuroblastoma development and increased tumor penetrance versus MYCN-only fish.
(A) Kaplan-Meier analysis of dβh-MYCN;RRM2 double transgenic zebrafish (left) and the mosaic model expressing cmlc2-eGFP/dβh-RRM2 in dβh-MYCN zebrafish (right) both show significant accelerated neuroblastoma formation and strongly increased tumor penetrance compared to dβh-MYCN fish. (B) Fluorescence microscopy images show the development of neuroblastoma tumors over time in MYCN-only (GFP) or MYCN-RRM2 double transgenic fish (mCherry). (C) RT-qPCR analysis showing human RRM2 overexpression in the dβh-MYCN;RRM2 double transgenic zebrafish compared to MYCN-only fish. Tdr7, looprn4, and hatn10 were used as housekeeping genes in this analysis. (D) H&E staining and immunofluorescent staining for the markers GFP, TH, and MYCN (×10 magnification). (E) Immunoblotting for S345pCHK1 and yH2AX for protein samples derived from MYCN and MYCN-RRM2 zebrafish. (F) Left: Time course analysis of replication stress markers in CLB-GA neuroblastoma cells upon prexasertib exposure by immunoblotting. Right: Quantification of the immunoblotting relative to vinculin. (G) Left: Time course analysis of replication stress markers in CLB-GA neuroblastoma cells before and after doxycyclin-inducible RRM2 overexpression by immunoblotting. Right: Quantification of immunoblotting relative to vinculin. (H) Signature score analysis of publicly available prexasertib sensitivity and resistance gene signatures in a large primary cohort of neuroblastoma cases (GSE62564). (I) Volcano plot showing the set of significantly up-regulated (red) and down-regulated (blue) genes in dβh-MYCN;RRM2 double transgenic versus dβh-MYCN fish. (J) GSEA of the gene expression profiles using the C5 curated MSigDB gene sets of dβh-MYCN;RRM2 double transgenic versus dβh-MYCN fish shows a significant up-regulation of DNA repair and genes related to cilium organization and movement, while down-regulated gene sets were predominantly related to synapse transmission gene sets [see volcano plot in (I) (blue)].
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