Fig. 6. TRMT61B abrogation diminishes the tumorigenic potential of ANE^high melanoma cell lines.

a Tumor growth in NSG mice injected with SK-MEL-103 cells consecutively transduced with lentiviral vectors harboring a luciferase reporter gene and a doxycycline inducible shRNA (iSH1) targeting TRMT61B expression. Monitoring tumor volume by external caliper indicates a statistically significant reduction in tumor growth after doxycycline administration (left). Two different experiments were carried out with 10 mice per group (doxycycline-treated and untreated). Representative macroscopic images and immunohistochemical staining of TRMT61B, Ki67, and cleaved caspase-3 in tumor sections from each mouse cohort are shown (right). Scale bars, 100 μm. b Representative bioluminescence images of doxycycline-treated and untreated NSG immunodeficient mice obtained 150 days after SK-MEL-103 tumor removal by surgical excision in order to facilitate metastatic spread. The color scale represents the bioluminescence levels (photons per second) emitted from the thoracic and abdominal region of xenografted mice (ventral view) ranging from low (blue), to medium (green) and high (red). The table below summarizes the incidence of metastatic lesions detected in both group of animals. Metastatic rates are calculated as the proportion of affected vs. total mice. c The Kaplan-Meier survival curve shows a reduced lifespan of Dox- mice in comparison with the Dox+ group. The log rank test was used to evaluate survival differences between both groups, and the HR indicates an increased risk of death in the untreated group. d Integrated density (at 1 day post injection, 1dpi) and proliferation ratio (at 6 days post-injection, 6dpi) of SK-MEL-28 cells (both Cas9/sgRNA and Cas9 alone, treated or not with doxycycline) injected into the circulation of the zebrafish embryos (top histograms). The red line at 6dpi represents a threshold below which cell proliferation stops. Integrated density at 1dpi is a total measure of the fluorescence of the fish tails. Average tumor number present in the tail of each condition tested at 1 and 6dpi is also shown (bottom histograms). n_replica = 20 embryos/condition (3 replica), n_total = 60 embryos/condition; Error bars represent standard error. e SK-MEL-28 cells containing CRISPR/Cas9 elements were subcutaneously injected in nude mice being treated or not with doxycycline from the very beginning to schedule a preventive strategy. As is shown in the graph, doxycycline administration causes a statistically significant tumor growth retardation. One experiment was performed with 12 animals per group. Error bars represent standard error in all the panels. *P < 0.05; **0.001 < P < 0.01; ***P < 0.001 (Student’s t-test; unpaired, 2-tailed).