Additional file 7 of Novel cellular systems unveil mucosal melanoma initiating cells and a role for PI3K/Akt/mTOR pathway in mucosal melanoma fitness
Creators: name: Monti, Matilde
name: Benerini Gatta, Luisa
name: Bugatti, Mattia
name: Pezzali, Irene
name: Picinoli, Sara
name: Manfredi, Marcello
name: Lavazza, Antonio
name: Vanella, Virginia Vita
name: De Giorgis, Veronica
name: Zanatta, Lucia
name: Missale, Francesco
name: Lonardi, Silvia
name: Zanetti, Benedetta
name: Bozzoni, Giovanni
name: Cadei, Moris
name: Abate, Andrea
name: Vergani, Barbara
name: Balzarini, Piera
name: Battocchio, Simonetta
name: Facco, Carla
name: Turri-Zanoni, Mario
name: Castelnuovo, Paolo
name: Nicolai, Piero
name: Fonsatti, Ester
name: Leone, Biagio Eugenio
name: Marengo, Emilio
name: Sigala, Sandra
name: Ronca, Roberto
name: Perego, Michela
name: Lombardi, Davide
name: Vermi, William
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Space Science
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: Pharmacology
subject: Chemical Sciences not elsewhere classified
subject: Cancer
subject: Hematology

description: Additional file 7: Figure S1. IHC analysis of SN-MM parental tumor biopsies and corresponding cell blocks (CB). Expression of melanocytic markers MITF, HMB45, MART, TYROSINASE, S100 and tumor associated fibroblast marker α-SMA. Sections are counterstained with hematoxylin. Parental tumors: Magnification 200X, scale bar 100 µm. CB: Magnification 400X, scale bar 50 µm. Figure S2. Scoring of melanocytic biomarkers in patient’s tumors, their corresponding SN-MM cell lines and cell-derived xenografts. Heatmap summarizing immunohistochemical expression of melanocytic biomarkers and α-SMA on the SN-MM cell lines cell block, patient’s tumor biopsies and mouse skin xenografts. Four-tiered scoring system was adopted, as described in methods section. NA: not assessed. Figure S3. Proliferation index and density of mitotic cells in SN-MM cell lines. A Representative images of ki67 and ph-H3 staining in SN-MM5 cell-block. Sections are counterstained with hematoxylin. Magnification 400X, scale bar 50 µm. B, C Quantification of ki67 and ph-H3 staining in SN-MM cell blocks. Histograms represent the percentage of positive cells out of total cells in three 20X field analyzed on digitalized slides. D Proliferation of SN-MM cell lines measured by MTS assay at 24 h, 48 h, and 72 h (n = 5). Histograms represent the absorbance at 490 nm that is directly proportional to the number of live cells in culture. One-way ANOVA statistical analysis and Bonferroni’s multiple comparison post-test have been performed. *p &lt; 0.05; ** p &lt; 0.01; *** p &lt; 0.001. Figure S4. Immunofluorescence staining of melanocytic biomarkers in SN-MM cell lines. SN-MM cells were cultured and stained as labeled. Cell nuclei were stained with DAPI (blue). Representative images of expression of MART (green), SOX10 (red) and S100 (green) are shown. Scale bar 20 µm. Figure S5. Ultrastructural features of SN-MM cell lines. TEM images showing cell morphology and heterogeneity among SN-MM cell lines (A–F); electron dense melanocytes are more evident in NHEM (F) and SN-MM5 (E) cell lines. Scale bars are 10 µm. Figure S6. Ultrastructural analysis of melanosomes on SN-MM cell lines. TEM images showing low electron dense pre-melanosomes in SN-MM1 (A), SN-MM2 (B) and SN-MM3 (E), early stage melanosomes presenting a fibrillar pattern in SN-MM2 (C, D), SN-MM3 (F, G), and SN-MM5 (I), and high electron dense mature melanosomes in SN-MM4 (H) and SN-MM5 (I). Scale bars are 1 µm (A, B, H, I), 500 nm (C, E, G) and 200 nm (D, F). Figure S7. Gains and losses of chromosome regions in five SN-MM cell lines. Overview of rearranged chromosomal regions in SN-MM1 (A), SN-MM2 (B), SN-MM3 (C), SN-MM4 (D), SN-MM5 (E), and SN-MM5.P1 (F) cell lines as detected by array-comparative genomic hybridization (aCGH). Red and green lines on the side of each chromosome represent losses and gains, respectively. Figure S8. IHC analysis of SN-MM parental tumor biopsies and corresponding cell-derived xenografts. Expression of melanocytic markers SOX10, MITF, MART, TYROSINASE, HMB45, and S100. Sections are counterstained with hematoxylin. Magnification 200X, scale bar 100 µm. Figure S9. NRAS mutation in SN-MM3. Sequence chromatograph of NRAS exon 2 of genomic DNA from SN-MM3 cell line (upper panel), parental tumor biopsies (middle panel), and cell-derived xenograft (lower panel) showing the c.35G&gt;A mutation resulting in NRAS p.Gly12Asp. Reverse sequence is shown. Figure S10. MIC and intermediate phenotypes in parental tumors and SN-MM-CDX from F1 generation. Sections are from parental tumor biopsies (A) and CDX obtained from SN-MM4 F1 generation (B), stained as indicated. Yellow rectangles mark cells co-expressing MITF and CD271 and lacking ZEB1 and CDH1. Green rectangles mark cells co-expressing ZEB1 and CD271 and lacking MITF and CDH1. Black arrows point cells co-expressing MITF and CDH1 and lacking ZEB1 and CD271. Magnification 40x, Scale bar 25 µm. Figure S11. Dose-response curves of cisplatin and temozolomide in SN-MM cell lines. SN-MM cells were treated with cisplatin (0.625–20 µM; A) for 72 h (SN-MM1; SN-MM2; SN-MM3; SN-MM5) or 120 h (SN-MM4) or with temozolomide (3.125–200 µM; B) for 72 h. Results are expressed as percentages of viable cells versus untreated cells ± SD of three independent experiments, run in triplicate. Two-way ANOVA statistical analysis and Bonferroni’s multiple comparison post-test have been performed. For concentrations starting from 20 µM to 0.625 µM vs. untreated cells (A); *p &lt; 0.05; ** p &lt; 0.01; *** p &lt; 0.001. Figure S12. Differentially expressed proteins and pathways among SN-MM cell lines and NHEM. A Plot showing the optimal number of five clusters of quantified proteins, for the k-means clustering, maximizing the average silhouette width. B Heatmap showing the scaled protein expression; unsupervised k-means clustering of proteins is applied highlighting groups of proteins with selective upregulation in five groups of samples, corresponding to different specific cell lines. C, D Histograms showing the numbers of up- and down-regulated melanosome’s proteins (C) and the ratios between up- and down-regulated proteins (D) in SN-MM5 compared to NHEM and other SN-MM cell lines, obtained from the STRING functional enrichment analysis. E, F Venn diagrams depict the number of positively and negatively enriched pathways among SN-MM cell lines. The number of positively (ES &gt; 0.25; E) and negatively (ES &lt; − 0.25; F) enriched pathways (p-val &lt; 0.05) in each SN-MM cell line compared to NHEM are reported in the histograms. The five-group Venn diagrams showed 15 up-regulated (ES &gt; 0.25; E) and 3 down-regulated (ES &lt; − 0.25; F) commonly enriched pathways in all SN-MM cell lines. Venn diagrams are realized by using jvenn at the following link http://jvenn.toulouse.inra.fr/app/example.html . Figure S13. Upstream Regulators in the SN-MM cell lines. Heatmap showing the predicted expression of upstream regulators (z-score) by IPA in the SN-MM cell lines compared to NHEM. Upstream regulators with a significant (p &lt; 0.05) and biological relevant activation (Z-score ≧ 2) or inhibition (Z-score ≦ -2) at least in one sample are shown. Significant and biological relevant upstream regulators are highlighted with labels showing the value of the Z-score. Not available data for an upstream regulator are indicated in light green color. Figure S14. Networks of upstream regulators in SN-MM cell lines. A–D The IPA upstream regulator MITF is significantly inhibited (z-score ≤ − 2) in four out of five SN-MM cell lines. E–H The IPA upstream regulator RICTOR is significantly activated (z-score ≥ +2) in four out of five SN-MM cell lines. Figure S15. Activation of Akt and sensitivity to PI3K inhibition in SN-MM cell lines. A Western blots showing Akt and phosporylated-Akt(Ser473, pAkt) and Akt(Thr308, pAkt) expression in SN-MM cell lines cultured in complete RPMI medium (Ctrl), after o/n starvation (1% FBS; Starv), and replenished with 10% FBS for 30 minutes (FBS). GAPDH is used as housekeeping control. B–D Dose-response curves of SN-MM cell lines treated with LY294002 chemical inhibitor (1 µM, 5 µM, 10 µM, and 100 µM) after 24 h (B), 48 h (C) and 72 h (D) (n = 5 for SN-MM1, SN-MM5; n = 4 for SN-MM2, SN-MM3; n = 3 for SN-MM4). Half maximal effective concentration (EC50) of LY294002 is reported for each SN-MM cell line at 24 h, 48 h and 72 h (E).