Fig. 3

Co-expression of TNFR1 and TNFR2 on melanomas is required for solTNF-induced MAPKi resistance. CRISPR/Cas9-mediated TNFR1, TNFR2 and CD271 knockout (KO) variants of Sk-Mel-37 cell line were generated. A The effectiveness of gene alterations was confirmed by flow cytometry. B KO cell variants were compared to the unaltered “wild-type” (WT) cell line for their ability acquire resistance to BRAFi and MEKi in response to rhTNF stimulation using the MTT assay. Experiments were performed in quadruplicates. Data shown represent mean values of quadruplicate tests and whiskers represent standard error. *p ≤ 0.05. C, D Sk-Mel-37 WT, TNFR1, TNFR2 and CD271 KO cell lines were cultured in the presence or absence of solTNF for 15 min. Subsequently, cells were evaluated for phosphorylated NF-kB [phos-NF-kB p65(S536)] and p38 MAPK (pT180/pY182) levels by flow cytometry. Staining examples (C) and summary of the data (D) are shown. Data are representative of three independent experiments