Figure 3.

BRAF mutation and its association with PD-L1 in PTC in vitro. (A,B) Basal expression of PD-L1 in PTC cell lines. Proteins were isolated from three PTC cell lines and immuno-blotted with antibodies against PD-L1, pMEK1/2, MEK1/2, pERK1/2, ERK1/2 and GAPDH. Western blots were quantified and data are presented as mean ± SD of three independent experiments (n = 3). (C,D) BRAF inhibition increases PD-L1 expression. BRAF-mutated PTC cell lines were treated with indicated doses of vemurafenib for 48 h. After cell lysis, equal amounts of proteins were separated by SDS-PAGE, transferred to immobilon membrane, and immuno-blotted with antibodies against PD-L1, pMEK1/2, MEK1/2, pERK1/2, ERK1/2 and GAPDH as indicated. Western blots were quantified and data are presented as mean ± SD of three independent experiments (n = 3). * Indicates a statistically significant difference compared to control with p < 0.05. (E,F) BRAF inhibition on cell growth. BRAF-mutated PTC cell lines were treated with indicated doses of vemurafenib for 48 h. Cells (5 × 10²) were then re-seeded into a 6-well plate, and grown for an additional 6–8 days, then stained with crystal violet and colonies were counted. Data presented in the bar graphs are the mean ± SD of three independent experiments (n = 3) which were repeated at least two times with the same results. * Indicates a statistically significant difference compared to control with p < 0.05.