Fig. 3.

Effects of MK2206, perifosine, PLX4032, or AZD6244 and their indicated combinations on cell cycle of thyroid cancer cells. A, OCUT1 cells were treated with 1 μm MK2206, 0.5 μm PLX4032, or 0.2 μm AZD6244, individually or in the indicated combinations, for 24 h. Cells were then collected, stained with PI, and subjected to flow cytometry. B, Effects of MK2206, PLX4032, and AZD6244 on the expression of cell cycle regulators. OCUT1 cells were treated with 1 μm MK2206, 0.5 μm PLX4032, and 0.2 μm AZD6244, individually or in the indicated combinations, for 24 h. Cell proteins were subjected to Western blotting, using antibodies against p-ERK, p-Akt, p27, p21, cyclin D1, or β-actin. β-Actin was used for quality control. Con, Control. C, OCUT1 cells were treated with 3 μm perifosine, 0.5 μm PLX4032, and 0.2 μm AZD6244, individually or in the indicated combinations, for 24 h and subjected to cell cycle analyses as in A. D, OCUT1 cells were treated with 3 μm perifosine, 0.5 μm PLX4032, and 0.2 μm AZD6244, individually or in the indicated combinations, for 24 h, followed by Western blotting analyses as in B. Relative quantification of p27, p21, and cyclin D1 shown below the corresponding bands was determined by the ratio of the density of the indicated proteins of different treatments to vehicle control treatments, which had already been divided by the density of their respective β-actin bands.