Fig. 6: Characterization of TG2 KO melanoma tumorigenic potential in vitro and in vivo primary tumors and metastatic formations.

A Colony formation assay with quantification of the number of colonies per sample (number of independent biological replicates = 5). The number of colonies was assessed with ImageJ (One-Way ANOVA, ^*p < 0.05). B Growth curve comparing the proliferation rate (expressed in growth percentage/hours) between B16F10 WT and the two TG2 KO clones. Parental B16F10 and Cas9-transfected cells displayed the same proliferation rate (not shown, number of independent biological replicates = 3). C–E Analysis of in vivo primary tumor growth from C57BL/6 WT orthotopic mice models after injection with the indicated cell lines. 4 animal models were injected for each group. Excised tumors are reported (C), showing a difference in pigmentation relatable to the different melanin content between TG2 WT and KO clones. Tumors were measured daily to assess the growth volume (D) and were weighted after the excision (E). F Analysis of lung experimental metastasis formation in C57BL/6 mice induced from B16F10 WT and TG2 KO tail vein injection. A picture of the front and back of mice lungs is reported for each condition. White arrows point to the experimental metastatic processes. G Multiplex IHC on lung experimental metastasis tissue of C57BL/6 mice induced from B16F10 WT and TG2 KO tail vein injection and relative tumor area quantification. Melanoma cells infiltration in tissues was visualized by anti-Melan-A staining (in yellow). DNA was stained with DAPI (in blue). Multiple 4 µm sections from 4 mice per conditions were used for the statistical analyses. Statistical analyses of three or more groups were performed with One-Way ANOVA. Two-way ANOVA with Bonferroni’s test was used to compare the data with two variables. Statistical significance is specified with asterisks (^*p < 0.05). Data are represented as mean ± SEM.