Fig 2: SOX10 knockout reduces expression of HVEM and CEACAM1(A) Parental mouse melanoma YUMM1.1 cells and Sox10 knockout clones were treated with or without 100 ng/mL IFN?, and HVEM expression was probed by flow cytometry staining. Mean fluorescence intensity (MFI) was quantified.(B) Parental human melanoma MeWo cells and SOX10 knockout clones were treated with or without 100 ng/mL IFN?. HVEM expression was probed by flow cytometry staining, and MFI was quantified.(C) Parental mouse melanoma YUMM1.1 cells and Sox10 knockout clones were treated with or without 100 ng/mL IFN?. CEACAM1 expression was probed by flow cytometry staining, and MFI was quantified.(D) Parental human melanoma MeWo cells and SOX10 knockout clones were treated with or without 100 ng/mL IFN?. CEACAM1 expression was probed by flow cytometry staining, and MFI was quantified.(E) CEACAM1 expression was probed by western blot in parental mouse melanoma YUMM1.1 cells and Sox10 knockout clones. The arrow indicates a nonspecific band ("n.s.").(F) CEACAM1 expression was probed by western blot in parental human melanoma MeWo cells and SOX10 knockout clones. All data in this figure represent 3 biological replicates; *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 3: Effects of HVEM or CEACAM1 alone on tumor growth(A) HVEM was expressed in YUMM1.1 parental and CR. #1.41 Sox10 knockout cells by lentiviral transduction. HVEM expression was validated by flow cytometry staining. Data are representative of 3 biological replicates. An unstained control is shown in light gray.(B) YUMM1.1 parental, CR. #1.41 Sox10 knockout cells, or HVEM-overexpressing cells were injected into BL6 mice, and tumors were measured by digital caliper every 2–3 days. Data were collected from 7 mice per group.(C) Related to (B), time-to-tumor onset was tracked. Tumors were considered fully formed when they reached ~50 mm3.(D) Two individual clones were generated from CRISPR-Cas9 knockout of Ceacam1 in YUMM1.1 cells. CEACAM1 surface expression was evaluated by flow cytometry. Data are representative of 3 biological replicates. An unstained control is shown in light gray.(E) YUMM1.1 parental, CR. #1.8, or CR. #1.30 Ceacam1 knockout cells were injected into BL6 mice, and tumors were measured by caliper every 2–3 days. Data were collected from 10 mice per group.(F) Related to (E), time-to-tumor onset was tracked. Tumors were considered fully formed when they reached ~50 mm3.**p < 0.01, ***p < 0.001.

Fig 4: SOX10 is correlated with CEACAM1 and HVEM expression in patient datasets(A) RNA-seq from the melanoma TCGA dataset was visualized using cBioportal, and the mRNA expression level of HVEM (TNFRSF14) was plotted versus SOX10 expression (Cerami et al., 2012; Gao et al., 2013). Shown are values batch normalized from Illumina HiSeq_RNASeqV2. SOX10 mutation type, structural variant, and copy number are indicated for each sample.(B) RNA-seq from the melanoma TCGA dataset was visualized using cBioportal, and the mRNA expression level of CEACAM1 was plotted versus SOX10 express ion (Cerami et al., 2012; Gao et al., 2013). Shown are values batch normalized from Illumina HiSeq_RNASeqV2. SOX10 mutation type, structural variant, and copy number are indicated for each sample.(C) Single-cell RNA-seq data of malignant cells (Jerby-Arnon et al., 2018) were visualized by t-distributed stochastic neighbor embedding (tSNE) plots by using the Single Cell portal (https://singlecell.broadinstitute.org/single_cell), whereby each cluster represents a distinct melanoma tumor and each circle represents an individual cell.