Supplementary Figure 5: Identification of melanosomal miRNAs and their trafficking into fibroblasts.

(a) Venn diagram showing the overlap between the 100 most highly expressed miRNAs in melanosomes, exosomes and melanoma-related miRNAs (literature based 41,42). (b) Yield of mature miRNAs in preparations from whole cells, mature melanosomes, and early melanosomes. Data were normalized to levels of RNU6B. Error bars represent ± SEM (n = 3 independent experiments). (c) Levels of indicated mature miRNAs were evaluated in fibroblasts treated with melanosomes or PBS (control) using RT-PCR. Data were normalized to RNU6B levels. Error bars represent ± SEM, ^∗ indicates p < 0.05 (n = 4 independent experiments). (d) Table of the five microRNAs that are most up-regulated in mature melanosomes compared to their levels in whole cells. (e) Expression level of mature mmu-miR-211 in human fibroblasts after treatment with B16F10 melanosomes was determined using qRT-PCR. Data were normalized to RNU6B (n = 2 independent experiments). (f) Human primary fibroblasts were transfected with human or mouse miR-211 mimic (hsa and mmu, respectively). qRT- PCR analysis of both miRNAs. Data were normalized to RNU6B (n = 2 independent experiments). (g) Mature miRNAs levels in normal and melanoma melanosomes (n = 2 independent experiments). (h) Levels of mature miRNAs in indicated fractions (n = 2 independent experiments). (i) Levels of mature miRNAs in secreted melanosomes treated with the inhibitor (SB202190), normalized to RNU6B (n = 2 independent experiments).