Fig. 4.

Fig. 4.

Effect of miR-204 modulation on melanoma incidence. (A–D) Characterization of melanoma tumors that develop in adult fish of the Tg(mitfa:BRAFV600E);p53-/-;mitfa-/- line, injected with miniCoopR-I vectors at 1-cell stage. (A) Schematic representation of the dox-treatment schedule used. (B) eGFP fluorescence is detectable only upon treatment with dox. eGFP fluorescence was detectable in two out of three fish tested, one of which is shown in the pictures. (C) Macroscopic appearance of a fish with melanomas (red arrows). (D) Microscopic features of a melanoma tumor that developed on the head and was excised 1 month after it became visible: H&E (upper), Fontana Masson (middle), anti-BRAFV600E (lower). Images were acquired with a 4x objective. Scale bar: 500um. (E) qRT-PCR quantification of mature miR-204 levels. 1 month after they became visible, tumors that developed on the tail were excised from n=3 fish injected with SCR vectors, n=3 fish injected with miniCoopR-I-sponge-204 vector and n=6 fish injected with miniCoopR-I-pre-miR-204 vector. In all these tumors, effective induction of the transgenic construct was confirmed as higher eGFP mRNA levels compared to a piece of tail that was collected from a non-induced fish and taken as baseline. (F) Melanoma-free survival curve (left) and percentage of animals with/without melanoma (right) upon injection of miniCooR-I-sponge-SCR and miniCoopR-I-sponge-204. (G) Melanoma-free survival curve (left) and percentage of animals with/without melanoma (right) upon injection of miniCooR-I-pre-miR-SCR and miniCoopR-I-pre-miR-204. In melanoma-free survival curves, week count started when the first pulse of 10 uM dox was administered, i.e. at 2 months of age. Statistically significant differences are indicated with asterisks: *P<0.05.