Figure 3. TR4 contributes to the repressive effects of MEK-162 in controlling murine pituitary tumor cell proliferation and hormone synthesis.

A., Knockdown efficiency of shRNA TR4 in AtT20 cells was evaluated by Western Blotting. B., Proliferation rates in AtT20 shRNA TR4 stable transfectants and nonsense control transfectants were compared following MEK-162 treatment (20 and 40 μM) for 72 h. The results are presented as fold change compared to vehicle treatment in nonsense control transfected cells. C., POMC promoter-luciferase reporter was transiently transfected into AtT20 shRNA TR4 stable transfectants and nonsense control AtT20 transfectants. 24 h later, cells were treated with MEK-162 for 48 h, and luciferase activities were measured and presented as fold change compared to vehicle treatment in nonsense control transfected cells. D., Ectopic expression of V5-tagged TR4 in AtT20 cells was confirmed by Western Blotting. E., AtT20 TR4 stable transfectants and vector control transfectants were treated with MEK-162 (20 and 40 μM) for 72 h to analyze cell proliferation rates. F., POMC promoter-luciferase reporter was transiently co-transfected with TR4 plasmid (200 ng and 400 ng) or vector control into AtT20 cells. 24 h later, cells were treated with MEK-162 in Opti-MEM for 48 h, and luciferase activities were measured and presented as fold change compared to vehicle treatment in vector transfected cells. G., After AtT20 TR4 stable transfectants and vector control transfectants were treated with MEK-162 (20 and 40 μM) for 24 h, POMC mRNA expression was determined by Q-PCR and presented as fold change compared to vehicle treatment in vector transfected cells. All the transfectants used were cell pools. Each bar indicates the mean ± standard error of triplicate tests. * p < 0.05; ** p < 0.01; ***p < 0.005. Data shown are representative of at least three independently performed experiments.