Figure 5.

Silencing of PD-L1 decreases cell growth of BRAF-mutated cell lines. (A,B) Knockdown of PD-L1 decreases clonogenicity. BRAF-mutated PTC cells were transfected with scrambled siRNA and two different PD-L1 siRNAs (50 nM). Forty-eight hours post-transfection, cells (5 × 10²) were 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 siRNA control with p < 0.05. (C,D) Knockdown of PD-L1 decreases AKT phosphorylation and downregulates the expression of anti-apoptotic proteins and induces the cleavage of caspase-3 and PARP. BRAF-mutated PTC cells were transfected with scrambled siRNA and two different PD-L1 siRNAs (50 nM). Forty-eight hours post-transfection, cells were lysed and equal amounts of proteins were separated and immuno-blotted with antibodies against PD-L1, pAKT, AKT, Bcl-2, Bcl-xL, caspase-3, cleaved caspase-3, PARP 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 siControl with p < 0.05. (E,F) Knockdown of PD-L1 caused no effect on MEK/ERK activation. BRAF-mutated PTC cells were transfected with scrambled siRNA and two different PD-L1 siRNAs (50 nM). Forty-eight hours post-transfection, cells were lysed and equal amounts of proteins were separated 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). * Indicates a statistically significant difference compared to siControl with p < 0.05.