Supplementary Figure 3: Transfer of melanoma melanosomes into fibroblasts leads to reprogramming.

(a) Tyrosinase activity of MNT-1 whole cell extracts or isolated mature and pre-mature melanosomes. Values are normalized to total protein concentration. Error bars represent ± SEM, ^∗ indicates p < 0.05 (n = 5 independent experiments). (b) Tyrosinase protein levels in indicated fractions. Rab27a was analyzed as a loading control. (c) Electron microscopy analysis of exosomes. (d) Expression of CD63 and CD81 in exosomes purified from MNT-1 cells by FACS analysis. Upper panel shows the analysis of expression of CD63 (blue) compared to unstained exosomes (red). Middle panel shows the analysis of expression of CD81 (blue) compared to unstained exosomes (red). Lower panel shows the analysis of expression of exosome markers CD63 and CD81 (blue) compared to unstained exosomes (red). (e) Protein levels in melanosomes isolated from MNT-1 cell homogenates and exosomes isolated from MNT-1 conditioned medium. Hsc70 was used as a loading control. (f) ND-1 levels in indicated fractions, normalized to GAPDH (n = 1 independent experiment). (g) Immunofluorescence staining of HaCat cells upon melanosome treatment. Melanosomal markers: tyrosinase, Tyrp1, and HMB-45 appear in yellow, red, and green, respectively. DAPI-stained nuclei appear blue. (h) Standard curves of melanosome number based on melanin and protein concentrations. (i) Fibroblasts migration upon osteopontin (400 nM) treatment. Unprocessed scans of Western Blots are shown in Supplementary Fig. 8.