Fernandez Del Ama et al., 2016 - Reprofiling using a zebrafish melanoma model reveals drugs cooperating with targeted therapeutics. Oncotarget   7(26):40348-40361 Full text @ Oncotarget

Fig. 1

Melanocyte hyplerplasia in V12HRAS-expressing zebrafish embryos. a. Upper pictures: WT (left) and V12RAS (right) transgenic adult fish. Lower pictures: 5dpf embryos. b. Expression of exogenous V12RAS detected by RT-PCR at different time points. β-catenin is a control for cDNA integrity. c. Quantification of the number of melanocytes in thirty wild-type (WT) or non-injected control (NIC), standard control oligonucleotide (STC)-injected or antisense oligonucleotide targeting exogenous V12RAS (V12RAS olig)-injected 5dpf V12RAS transgenic embryos. Shown are means ±SEM. d. Quantification of the melanin absorbance (λ= 340nm) for eight wild-type (WT) or NIC, STC-injected and V12RASolig-injected 5dpf V12RAS transgenic embryos. Shown are means ±SEM for six independent experiments. (c) and (d) For easier reference, the dashed line indicates the mean value for wild-type embryos. *** P<0.001 independent samples t-test.

PHENOTYPE:
Fish:
Observed In:
Stage Range: Day 5 to Adult

Fig. 2

Molecular characterization of the V12RAS model. a–d. Transverse sections of V12RAS larvae. (a) H&E staining. Scale bar = 0.2 mm. (b–d) IHC on bleached sections. (b) Non-specific primary control, (c) phospho-ERK and (d) phospho-Akt. Positive stain (brown) denoted by asterisks. e. Results of immunoblotting protein extract (30 μg) from 30 pooled 5-dpf embryos exposed for 4 h to the specified drug at the indicated concentrations. Representative immunoblots are depicted with densitometric quantification shown immediately below (mean p-protein/total protein ± SEM for three independent experiments). *P<0.05 compared to vehicle control; independent samples t-test.

Fig. 3 ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

Fig. 5

Synergistic effect of Rapamycin. a. In-vivo drug dose curve for Rapamycin using the melanin assay. Rapamycin was tested at different concentrations in V12RAS and WT embryos, and in combination with MEK and mTOR/PI3K inhibitors in the transgenic embryos. Points depict mean ± SEM for three independent experiments. b. Imaging-based method for determining melanocyte burden. Values shown are mean pigmented areas ± SEM for thirty embryos *** P<0.001 independent samples t-test. c. Melanocyte area quantification in V12RAS embryos treated with PD184352 (PD) (0.1 μM), NVPBEZ235 (NVP) (0.3 μM), Rapamycin (1 μM), Rapamycin (1 μM) in combination with PD (0.1 μM), and Rapamycin (0.25 μM) in combination with NVP (0.3 μM). *** P<0.001 One-way ANOVA. d. Lateral views of 5dpf zebrafish embryos from the yolk sac to nearly the end of the tail treated as indicated. Average cell number (%) measured in e. WM1361 and f. A375 cell lines when treated with different combinations of Rapamycin and NVPBEZ235 concentrations for 72h. The colour code corresponds to the Chou-Talalay combination index (CI) used to quantify the level of synergism. g. The corresponding IC50 values for the above cell line treatments.

Acknowledgments:
ZFIN wishes to thank the journal Oncotarget for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ Oncotarget