Fig 2: Long-term proliferation of hPSC-derived melanocytes. (a) Doubling time measurements during the growing phase of the Mel-hESC and Mel-hiPSC, from passage 3 (P3) to passage 26 (P26). (b) Cumulated cell expansion calculated from passage 3 (P3) to passage 15 (P15). For the next parts of the figure, Mel-hPSC early passages (Mel-hPSC-E) are defined from P3 to P15 and Mel-hPSC late passages (Mel-hPSC-L) are defined from P15 to P26. (c) Microscopy analysis and immunofluorescence analysis of MITF, TYRP1, and PMEL17 in Mel-hESC-L and Mel-hiPSC-L. Scale bar is 50 µm. (d) Flow cytometry analysis of TYRP1 in Mel-hESC-L and Mel-hiPSC-L. Mel-hESC-L value represents the mean ± SD 0.05 of three independent experiments. (e) Expression of PAX3, MITF, and TYROSINASE proteins by Western blot in Mel-hESC-L and Mel-hiPSC-L compared to HEMs. Band intensities are normalized to β-actin. (f) AmpliSeq Cancer Hotspot Panel Library v2 in Mel-hESC and Mel-hiPSC at early and late passages. Samples tested included melanoma cell line (SK-MEL-28) as a positive control. The SNPs detected are compared to the database FATHMM (http://fathmm.biocompute.org.uk, accessed on 6 March 2017). (g) Soft agar assay for colony formation on early and late passages of Mel-hESC and Mel-hiPSC. SK-MEL-28 is used as a control for the melanoma cell line. HEMs are used as a control for adult melanocytes (scale bar is 50 µm).

Fig 3: Characterization of hPSC-derived melanocytes. (a) Diagram of the melanocytic differentiation from hPSCs. Illustrated using Servier Medical Arts-SMART image bank. (b) Microscopy images at different stages of melanocytic differentiation from hPSCs (scale bar: 50 µm). (c) Microscopy images of Mel-hESC and Mel-hiPSC at passage 6 (P6) compared to HEMs (P6). Immunofluorescence staining of MITF and TYRP1 in Mel-hPSC (scale bar: 50 µm). (d) Flow cytometry analysis of TYRP1 in Mel-hESC (P7), Mel-hiPSC (P6), and HEMs (P6). The Mel-hESC value represents the mean of six independent experiments with an SD ± 0.4. The HEM value represents the mean of three independent experiments with an SD ± 0.1. (e) Hierarchical clustering of gene expression. Comparison between HEMs (P6), Mel-hESC (P4), and hESC (n = 3). (f) Venn diagram showing comparative gene expression profiles of Mel-hESC (P4) and HEMs (P6). Gene lists are defined as genes upregulated in the two cell lines greater than 2-fold and with an FDR < 0.001 compared to hESCs. i represents common list between Mel-hESC and HEMs, ii as the 569 uregulated genes specific to Mel-hESC and iii as the 314 genes specific to HEMs. (g) Scatterplot showing a high correlation of 1921 gene expression levels in HEMs and Mel-hESC (R = 0.967) from indicated regions (i) of Venn diagram defined in (f).

Fig 4: CICD does not trigger proliferation of melanoma cancer cells. a Working model for testing the effect of apoptotic and CICD conditioned media on the proliferation of neighboring cells. b-c Incucyte analysis for the proliferation of WM115 H2B-mCherry cells grown in the presence of APO (b) or CICD (c) conditioned media, obtained 24 h after triggering either apoptosis or CICD. n = 4–5 independent experiments; mean values +/− s.e.m. d Same as in (b-c), while this time cell proliferation was assessed by quantifying the optical density (O.D.) of methylene blue staining of cells grown in either apoptotic or CICD media. e Representative images of methylene blue staining. f Compensatory proliferation tested and quantified by clonogenic survival assay performed using the same conditions described in (b). g Parental WM115 cells were incubated with doxycycline (1 μg/ml) and the conditioned media was then added on H2B-mCherry expressing WM115 cells while clonogenic survival was assessed. Actinomycin D (ActD) treatment was used as negative control. h Incucyte analysis for the cell death induction (SYTOX Green exclusion) triggered by CICD conditioned media in WM115 H2B-mCherry cells. Actinomycin D treatment (1 μM) is used as positive control for cell death induction. A representative experiment is shown. i Immunoblotting for ZEB-1-MITF transcription factors axis of WM115 cells grown in APO and CICD conditioned media for 48 h. Actin was used as loading control

Fig 5: Mitf expression is promoted by mutated SASH1 in vitro and in epithelial tissues affected by the Y551D-SASH1 mutation. (A) Exogenous SASH1 is not associated with endogenous Mitf in 293T cells. GFP-SASH1 was transfected into 293T cells. At 48 h following transfection, transfected cells were lysed, GFP-SASH1 was immunoprecipitated, and the associated endogenous Mitf was analyzed by IP-WB analyses. (B) Expression of endogenous Mitf was induced by Y551D SASH1. Exogenous Y551D SASH1 and a wild-type SASH1 were introduced into 293T cells. Following transfection, transfected cells were lysed and subjected to western blot analyses. **P<0.001 vs. wild-type SASH1 and ***P<0.001 vs. blank control. (C) In the lesional epithelial tissues of Y551D SASH1-affected individuals, SASH1- and Mitf-positive cells were demonstrated in different epithelial layers of the affected epithelial tissues and calculated and analyzed statistically. **P<0.01, ***P<0.001. Upregulation of SASH1 and enhanced Mitf were also induced in the affected tissues. Magnification, ×40. Mitf-positive cells, which were stained dark brown in the nucleus, are indicated by red arrows. (D) More melanin was synthesized and present in different epithelial layers of the affected skin epithelial tissues. Magnification, ×40.