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.