Fig 2: BRAF mutation modulates the expression of immune regulatory molecules. A The effect of IFN-γ treatment on the survival of tumor cells analyzed by flow cytometry. B, C The basal and induced expressions of PD-L1 in BRAF WT and mutant MC38 cells detected by flow cytometry and qRT-PCR. D The basal and induced expressions of MHC class I in BRAF WT and mutant cells detected by flow cytometry. E–G The basal and induced expressions of Psmb8, H2-D1 and B2M in BRAF WT and mutant cells detected by qRT-PCR. Cells were treated by 20 ng/mL IFN-γ for 48 h. Three independent replicates were performed for above experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Fig 3: D594A mutation confers lower aggressiveness on MC38 cells. A Immunoblotting of BRAF level in WT and mutant MC38 cells. Actin serves as an internal reference. B Representative immunoblotting of MEK, pMEK, ERK1/2, pERK1/2 and Cyclin D1 in the starvation group (right) and stimulation group (left). Cells were exposed to EGF (100 ng/mL) for 15 min after starvation for 36 h in stimulation group. C Proliferation ability of BRAF WT and mutant MC38 cells at 24 h, 48 h and 72 h detected by CCK-8 assay. D Detection of apoptosis by annexin V/PI staining after induction of FCCP for 36 h. E Growth ability of BRAF WT and mutant MC38 cells analyzed by clone formation assay. F, G Migration and invasion abilities of BRAF WT and mutant MC38 cells analyzed by transwell assays, respectively. Three independent replicates were performed for above experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Fig 4: DUSP KD leads to a suppression of MITF-dependent gene expression.(A) mRNA levels of MITF and its related genes were analyzed by RT–qPCR 48-h post-transfection and were referred to the expression level of siNT cells. Data are normalized to GAPDH and represent mean of three independent experiments. Statistical significance was calculated between siNT versus NT+T and siDUSP4 and siDUSP4 versus siDUSP4+ Tram. (B, C) Cells were transfected with siRNA against nontargeting control and DUSP4, DUSP6, DUSP10. (B, C) Forty-eight hours later, cell lysates were analyzed by Western blot (B), and DUSP10 mRNA levels were measured by RT–qPCR (C). Data are mean ± SEM, n = 2. (D) BRAF-mutant cell lines from different tissues were transfected with siRNA against DUSP4 and BRAF. After 48 h, cell lysates were analyzed by immunoblot. (E) MITF protein levels were assessed in the indicated BRAF-mutant cell lines by immunoblot.

Fig 5: Oncogenic BRAF fusion gene detected in MFS. a Schematic presentation of the proteins encoded by SLC37A3, BRAF, and SLC37A3-BRAF fusion gene. MFS major facilitator superfamily, RBD RAS-binding domain, C1 phorbol esters/diacylglycerol-binding domain (C1 domain), Kinase protein kinase. b BRAF fusion induced ERK activation. Proteins extracted from cells transfected with retroviruses containing control, BRAF-V600E, and SLC37A3-BRAF were immunoblotted with antibodies against BRAF, phosphorylated ERK (p-ERK), ERK, and β-Actin. c Tumorigenicity of SLC37-BRAF fusion. Representative images of mice subcutaneously transplanted with NIH3T3 cells expressing empty vector or the SLC37A3-BRAF fusion. NIH3T3 cells transformed with BRAF V600E were used as a positive control