Extended Data Fig. 7: DCC-derived cell lines reflect the diversity of ex vivo DCC phenotypes.
a, Expression of DCC-derived marker genes for the different phenotypes of early and late transitory (ET, LT), neural crest-like (NC), undifferentiated (U), and melanocytic (M) phenotypes in MelDCC 1-13, analyzed by bulk RNAseq in duplicates or triplicates of consecutive passages. b, Flow cytometric analysis of MelDCC 2, 5a, 6, 8, 10a, and 11 for NGFR (neural crest), Melan A (melanocytic), AXL (invasiveness), and MCSP expression. MelDCC2 was stained with anti-MCSP-FITC (clone EP-1), all other MelDCC lines with anti-MCSP-PE (clone 9.2.27). c, Western blot of 100 K sEVs or size-exclusion chromatography-separated 100 K sEV vesicle (SEC-V) and protein (SEC-P) fractions. Markers for exomeres (ACLY), and non-EV contaminants (fibronectin, FN1; calnexin, CANX; histone H2A; albumin, ALB) and sEV (CD81, TSG101), and pan-EV (HSP70, GAPDH) were used. Loaded per lane: 2.5×106 sEVs, 106 cells, 10 µg whole cell lysate (L). The albumin signal in SEC-V and SEC-P samples is due to the absence of a washing step in the SEC workflow, unlike ultracentrifugation. d, Particle size and NTA quantification of sEVs secreted per million MelDCC cells over 48 h. MelDCC 2, 5a, 6, 8, 10a, and 11 (n = 18, 15, 9, 12, 11, and 23). N = biological replicates. e, Size distribution of CD81+ 100 K sEVs from MelDCC 10a (n = 7 biological replicates, indicated by different color codes) determined by NTA. f, Western blot of 100 K sEVs from untreated or IFNG-treated MelDCC 6 for 4 weeks. Loaded are sEVs from 0.5×106 cells/lane.
