Fig. 4.

TBK1 is a mitotic kinase involved in PLK1 activation. (A) TBK1 is activated in a cell-cycle-dependent manner. A549 cells were cell-cycle-synchronized by double thyimidine block (DTB) and released into fresh medium, and then whole cell extracts were prepared at the indicated times. Cell-cycle stages were verified by cyclin B1 and phospho-histone H3 (Ser-10) immunoblotting. *Nonspecific. (B) TBK1 directly phosphorylates PLK1. TBK1 in vitro kinase experiments were performed by using recombinant TBK1 (420 ng) and immunopurified Myc-tagged-kinase dead PLK1 (K82R) from 293FT cells as a substrate. (C) Impaired mitotic phosphorylation of PLK1 by TBK1 knockdown. Cells were infected with control or shTBK1 lentiviruses and then exposed to nocodazole (50 ng/mL) the next day for 18 h. (D) Analysis of correlation between TBK1 and PLK1 sensitivity in lung cancer cells. Nine non-small-cell lung cancer (NSCLC) cell lines were exposed to two different PLK1 inhibitors, BI6727 and BI2536 (0.5 µM, 72 h), or infected with control or shTBK1 lentiviruses (6 d), and then cell viability was examined. Each cell line’s remaining cell viability compared with control was plotted, and correlation coefficient (R²) was calculated.