Figure 2: Inhibition or silencing of TBK1 induces mitotic defects and inhibits mitosis.

(a) Knockdown of TBK1 by transfecting two different siRNAs in A549 and H1650 cells resulted reduces the number of mitotic cells. Control siRNA refers to transfection with a non-targeting siRNA, (b) Knockdown of TBK1 using lentiviral shRNA in H1650, H460 and A549 cells reduces the number of mitotic cells, For a,b, error bars represent standard deviation (s.d.) *P<0.05; t-test was applied. Experiments were repeated three times. (c) Western blot analysis of cells synchronized at G1/S by double-thymidine treatment and released for 2, 4, 6, 8 or 12 h; phospho-TBK1, Phospho- S172 TBK1; TBK1, total TBK1; pH3S10, histone H3 phosphorylated at serine 10 (mitotic marker). (d) Western blot showing the levels of Histone H3 phosphorylated at serine 10 after TBK1 inhibition by BX795 treatment (0.5 μM) in H1650 and A549 cells. Cells were synchronized to G1/S by double-thymidine block and subsequently allowed to proceed to mitosis. R0, synchronized by double-thymidine block; R4, released for 4 h, R9 released for 9 h. (e) Western blot showing the levels of Histone H3 phosphorylated at serine 10 (H3pS10) after TBK1 depletion by siRNA transfection in A549 cells. (f) Western blots showing the levels of phospho histone H3 S10 (pH3S10), to test if overexpression of PLK1 can overrideTBK1 depletion. For this, A549 cells were transfected with control or TBK1 siRNA along with a control plasmid (pcDNA3) or mEmerald-PLK1 to overexpress PLK1. Following transfection, cells were subjected to double-thymidine block and then released to enter mitosis. Levels of pH3S10, TBK1 and PLK1 were assessed by western blotting. Control siRNA-treated cells showed elevated levels of pH3S10 when released from G1/S block, indicating entry into mitosis; in contrast, TBK1-depleted cells did not show high levels of pH3S10 even when PLK1 was overexpressed, suggesting PLK1 cannot rescue mitotic arrest triggered by TBK1 depletion.