Fig. 2

BRAF V600E promotes POMC promoter activity and ACTH production. a Immunoblotting analysis of p-Erk1/2 and total Erk1/2 in AtT-20 stably infected with wild-type BRAF or BRAF V600E. b Immunohistochemical study of p-Erk1/2 in tumor samples according to the mutational status of BRAF. Scale bars, 20 μm. P-value was calculated using two-tailed Fisher’s exact test (P = 0.036). c Immunoblotting analysis of indicated proteins in AtT-20 cells as described in a. d Activities of wild-type or various mutated POMC promoters in cells transfected with wild-type BRAF or BRAF V600E together with a control vector expressing renilla luciferase. Y-axis: ratio of luciferase activity/renilla activity, presented as the means of three experiments and expressed relative to empty control vector. Error bars represent SEM of three measurements. e Relative mRNA levels of POMC in AtT-20 cells stably infected with indicated constructs. Data are expressed relative to the empty control vector after normalization to Hprt. Error bars represent standard SEM of three measurements. *P < 0.05, **P < 0.01, two-tailed Student’s t-test (d, e). f The inhibitory effect of vemurafenib on ACTH production in primary cultured human corticotroph adenomas (two wild-type and two BRAF V600E). ACTH levels in culture media were measured by radioimmunoassay and presented as % of the control media. Error bars indicate SEM of three replicates. The dashed line represents IC₅₀