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1 DOI: /ncb2535 Figure S1 SOX10 is expressed in human giant congenital nevi and its expression in human melanoma samples suggests that SOX10 functions in a MITF-independent manner. a, b, Representative pictures of immunostaining for SOX10 in 3 subgroups of human congenital nevi (group I, II and III). 17 samples of giant congenital nevi from different human patients were divided into three subgroups based on the size of the congenital nevi: group I (1.5 to 10 cm), group II (11 to 20 cm) and group III (21 to 40 cm). Note the expression of SOX10 in differentiated melanocytes residing in the epidermis, while epithelial cells of the epidermis do not express SOX10 (epidermal-dermal border is demarcated by dashed lines). c, d, Expression analysis of SOX10 and MITF in human melanomas. TMA were scored based on the percentage of SOX10- positive and MITF-positive nuclear staining in melanoma cells and samples were divided into four groups with expression in less than 10% of tumor cells (group I), from 10 to 50 % (group II), from 50 to 90% (group III), and in more than 90% (group IV) of all tumor cells. Insets show high magnification views. c, Analysis of SOX10 and MITF expression in primary human melanomas. Two representative tumors immunostained for SOX10 and MITF from TMA of primary melanoma samples demonstrating that in 11% of cases SOX10 is expressed at grade IV, while MITF expression is evaluated as grade I (n=39 tumors). d, Analysis of SOX10 and MITF expression in metastatic TMA showing that SOX10 expression does not correlate to MITF expression in 16% of all cases analyzed (n=119 tumors). These data demonstrate that SOX10 might have a MITF-independent function in a subset of human melanomas. M, melanocytes, TMA, tissue microarray, BF, bright field; Scale bars, 50 mm. 1
2 Figure S2 Sox10 haploinsufficiency prevents skin hyperpigmentation, expansion of neoplastic cells, and melanoma formation in Tyr::Nras Q61K INK4a -/- Sox10 LacZ/+ mice. a, Pictures of Tyr::Nras Q61K INK4a -/- and Tyr::Nras Q61K INK4a -/- Sox10 LacZ/+ littermate mice at different time points. At postnatal day 12 (a, left panels) and at 5 months of age (a, right panels), Sox10 haploinsufficiency counteracts hyperpigmentation of paws and snout in Tyr::Nras Q61K INK4a -/- mice. Moreover, also in Tyr::Nras Q61K INK4a - /- mice, Sox10 haploinsufficiency resulted in formation of a belly spot (a, lower left panel). Note that Tyr::Nras Q61K INK4a -/- Sox10 LacZ/+ mice have pigmented hairs indicating normal melanocyte stem cell function. Scale bars, 5 mm. b, Pictures of Tyr::Nras Q61K INK4a -/- and Tyr::Nras Q61K INK4a - /- Sox10 LacZ/+ mice showing two examples of Tyr::Nras Q61K INK4a -/- mice with melanoma on the back skin (melanoma is demarcated by dashed lines) and their Tyr::Nras Q61K INK4a -/- Sox10 LacZ/+ littermates displaying no signs of melanoma. Please note that there is no hair greying in mice of both genotypes. Scale bars, 5 mm. c, S100 immunohistochemistry of skin sections from Tyr::Nras Q61K INK4a -/- and Tyr::Nras Q61K INK4a -/- Sox10 LacZ/+ mice. To decrease hyperpigmentation in the dermis, sections were bleached. Note rare example of few S100-positive cells associated with HFs in Tyr::Nras Q61K INK4a -/- Sox10 LacZ/+ mice and the presence of S100-positive glial cells in close proximity to the SG in both genotypes. Two examples for each genotype are shown. Scale bars, 50 mm. d, Immunostaining for Dct (green) in skin sections from Tyr::Nras Q61K and Tyr::Nras Q61K Sox10 LacZ/+ mice at postnatal day 9. Note the presence of multiple ectopic Dctexpressing cells in Tyr::Nras Q61K mice (indicated by arrows). Strikingly, Sox10 haploinsufficiency completely abolishes the presence of these Dct-positive cells in the dermis. Scale bars, 20 mm. HF, hair follicle, SG, sebaceous gland. 2
3 Figure S3 Characterization of SOX10 expression upon SOX10 knockdown in vitro in human melanoma cell lines. a, Western blot analysis of SOX10 expression in M and A375 human melanoma cell lines upon SOX10 knockdown after 48 and 96 hours showing that SOX10 levels are efficiently reduced as compared to the control samples. For SOX10 knockdown, shrna expression plasmids based on psuper-neo-gfp vectors (SOX10 shrna; scrambled (scr) shrna used as a control) were electroporated into human melanoma cell lines and samples were collected after 48 and 96 hours. b, c, Immunostaining for SOX10 (red) combined with GFP signal showing that in cells electroporated with scr shrna all GFP-positive cells express SOX10, while GFP-positive cells electroporated with SOX10 shrna show no SOX10 expression. Representative examples from A375 melanoma cell line (b) and from M cell line (c) are shown. Scale bars, 20 mm. Uncropped images of blots are shown in Supplementary Fig. S6. 3
4 Figure S4 SOX10 knockdown in human melanoma cell lines leads to decreased expression of melanocytic genes, increase of mesectodermal differentiation, increased apoptosis and deregulation of cell cycle. a, Venn diagram depicting the number of differentially upregulated or downregulated genes in human melanoma M cell line upon SOX10 knockdown after 48 and 96 hours. b, mrna expression of genes of melanocytic lineage after 96 hours of SOX10 knockdown. n=3 independent experiments, each carried out in triplicate. c, mrna expression of mesectodermal lineage genes after 96 hours of SOX10 knockdown. n=2 independent experiments, each carried out in triplicate. d-f, mrna expression of genes involved in the regulation of cell cycle in M cell line after 48 and 96 hours of SOX10 knockdown. n=3 independent experiments, each carried out in triplicate. Data represent the fold change as mean ± s.d. and are normalized to the control. 4
5 Figure S5 Knockdown of SOX10 induces cell cycle arrest and leads to increased apoptosis in human melanoma cells. a-b, FACS analysis of apoptosis in A375 cell line upon SOX10 knockdown. For apoptosis analysis, GFP-positive cells were gated (a) and Annexin V-positive cells were analyzed after 48 hours (b, left panel) and 96 hours (b, right panel) of SOX10 knockdown. c, d, Immunostaining for activated caspase-3 and quantification of activated caspase-3-positive cells in M cell line upon SOX10 knockdown after 96 hours. Percentage of caspase-3-positive cells per DAPI-positive nuclei was counted (n=400 DAPI-positive nuclei in each independent experiment). n=3 independent experiments. Data are presented as mean ± s.d. These data show that SOX10 knockdown influences survival of melanoma cells. e-g, FACS analysis of cell cycle in the A375 cell line upon SOX10 knockdown. GFP-positive cells were gated (e) and subjected further to PI selection gating. Resulting FACS profiles of A375 control and A375 SOX10 shrna cells at 48 hrs (f) and at 96 hrs (g) are presented. h, Quantification of cell cycle phase distribution. Percentage of cells in G1/G0 and S+G2 phases in M and A375 cell lines upon SOX10 knockdown after 48 and 96 hours are presented. n=3 independent experiments. Note that in control A375 cells, more cells are in S+G2 phase than in G1/ G0 phase. Data are presented as mean ± s.d. The data show that SOX10 knockdown results in cell cycle arrest in melanoma cells. hrs, hours, Scale bars, 50 mm. 5
6 Figure S6 Full length blots from Supplementary Fig. 3a. 6
7 Table S1 Clinical data of human patients diagnosed with giant congenital melanocytic nevi. 17 samples of giant congenital nevi from different human patients were divided into three subgroups based on the size of the congenital nevi: group I (1.5 to 10 cm), group II (11 to 20 cm) and group III (21 to 40 cm) and analyzed for SOX10 expression. CMN, congenital melanocytic nevus, f, female, m, male. 7
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