Fig. 6.

BRAF V600E promoted FOS binding to GABPB by upregulating FOS phosphorylation. a Western blotting analyses for phosphorylated-FOS (p-FOS), total FOS (t-FOS), BRAF, and beta-actin in K1 and A375 cells with or without stable BRAF knockdown. b Western blotting analyses of p-FOS, t-FOS, p-ERK, and beta-actin in K1 and A375 cells treated with 0.5 μm PLX4032 for 0, 1, and 24 h. c, d, e Effects of FOS phosphorylation on GABPB and TERT activation. c KAT18 cells were serum-starved for 24 h and transiently transfected with FOS wild-type (FOS-wt) or mutant (FOS-mut) bearing none of the potential ERK-targeted phosphorylation sites, followed by western blotting analysis for TERT, GABPB, p-FOS, t-FOS, and beta-actin. d KAT18 cells were transfected with FOS-wt or FOS-mut along with GABPB 5′-UTR luciferase reporter and Renilla luciferase (pRL-TK) plasmids in the absence of serum for 24 h, and the luciferase activities were then measured. e KAT18 cells were transiently transfected with FOS-wt or FOS-mut, together with TERT promoter luciferase reporters harboring the C228T or C250T mutation, and pRL-TK for 24 h, followed by luciferase assays. f ChIP assay for FOS binding to the 5′-UTR of GABPB in K1 and A375 cells. g Western blotting analyses for GABPB, p-FOS, p-ERK, and t-ERK in the parental and BRAF-V600E knock-in SW48 cells. h ChIP assay for FOS binding to the 5′-UTR of GABPB in SW48 cells. i GABPB 5′-UTR region-luciferase reporter assays in SW48 cells. j Wild-type (WT) BRAF and BRAF V600E were stably introduced to express in WRO cells, followed by western blotting analyses of the expression of GABPB, p-FOS, BRAF, p-ERK, and t-ERK after serum starving overnight. **P < 0.01, ***P < 0.001, by two-tailed Student’s t test. n.s. not significant. All values represent the average ± standard deviation (SD) of triplicate samples and similar results were obtained in two additional independent experiments