Figure 6: TBK1 depletion alters microtubule dynamics.

(a) Shcontrol A549 or H1650 cells treated with 0.1 μM nocodazole for 15 min showed complete disruption of microtubules, while TBK1-depleted shTBK1 A549 or H1650 cells retained stable microtubules as seen by staining for alpha tubulin (green) and CEP170 (red). (b) Mitotic spindles in TBK1-depleted A549 and H460 cells were more stable after nocodazole treatment as compared to control cells. Alpha tubulin (green) shows mitotic spindles. (c) shTBK1 A549 or H1650 cells when subjected to cold temperature (4 °C) and subsequent incubation at warm temperature (37 °C) had stable microtubules versus analogously treated shcontrol A549 or H1650 cells. Alpha Tubulin (green), CEP170 (red) and DNA (DAPI, Blue). (d) Similar results were observed in mitotic cells where mitotic spindles of TBK1-depleted A549 or H460 cells were more stable compared to control shRNA-treated cells. Alpha tubulin (green). (e) Stability of microtubules were drastically reduced in A549 cells overexpressing TBK1 when treated with 5 μM nocodazole and allowed to regrow for 7 min; Scale bar, 10 μm for e. (f) Low magnification images of cells in c. Scale bar, 25 μm. (g–i) TBK1 is required for the binding of CEP170 with Kif2b (g) as well as for the association of NuMA with dynein (i), as seen by immunoprecipitation–western blot analysis. (h) Western blot for pH3S10 to confirm mitotic fractions.