Additional file 1: Table S1. Melanoma cell line phenotypes and MAPKi sensitivities. Table S2. Melanoma patient characteristics. Figure S1. BRAFV600E+ melanoma cell lines express various levels of mutant BRAF protein. PCR-generated BRAFV600E mutation status of human melanoma cell lines utilized in this study was confirmed by flow cytometry (BRAFV600E Antibody VE1; Roche). Figure S2. Correlation of TNFR1, TNFR2 and CD271 expression levels on BRAFV600E+ melanoma cell lines. Expression levels of the three receptors adjusted for respective IgG controls (Fig. 1) were evaluated by linear correlation. Figure S3. Generation and activation of monocyte-derived macrophages. Human monocyte-derived macrophage (MΦ) phenotype was confirmed by A) flow cytometry (CD14+CD68+CD80+CD86+) and B) their ability to release solTNF and VEGF in response to LPS + IFNγ activation. Figure S4. TNFR2+ primary BRAFV600E+ melanoma cell line can acquire resistance to BRAFi in response to solTNF treatment. (A) Primary melanoma cell lines were generated from two BRAFV600E+ melanoma patient biopsies. Their TNFR1, TNFR2 and CD271 profiles were evaluated by flow cytometry. (B) Early passage cell lines (10 passages or less) cultured in the presence or absence of solTNF were tested for their sensitivity to BRAFi and MEKi-mediated cytotoxicity. Data shown represent mean values of quadruplicate tests and whiskers represent standard error. *p ≤ 0.05. Figure S5. TNFR2 expression on BRAFV600E+ melanoma is upregulated in response to IFN-γ, but not MAPKi. SK-Mel-28 cell line was cultured for 48 h in the presence or absence of IFN-γ (1000 IU/ml), 0.5 µM BRAFi (0.5 µM) and MEKi (0.2 µM). Subsequently, cells were collected and stained for TNFR2 by 2-step staining as previously described. Data shown are representative of three independent experiments.
