TTK inhibition overrides the SAC mediated cell cycle arrest and leads to aberrant cell cycle progression, multi-nucleation and apoptosis.
(A) Immunoblot of HPAC and PANC-1 PDAC cell lines arrested in mitosis by treatment with nocodazole. Cells were then treated with 2 μM AZ3146 for 4 h and probed for expression of cyclin B1. (B) Representative flow cytometry plots of the cell cycle of HPAC and PANC-1 cell lines of 2 experiments. Cells were transfected with control or TTK targeted siRNA. 72 h post transfection cells were fixed and stained with propidium iodide. DNA content was assessed by flow cytometry. (C) Quantitation of B showing distribution of cells in each phase of the cell cycle. (D) Confocal microscopy of the PANC-1 cell line stably expressing a GFP-Histone 2B construct to visualize DNA. Chromosomal instability is visible in cells depleted of TTK in the form of multi- and micro-nucleation. (E) Quantitation of cells with multi- or micro-nucleated phenotypes. (F) Scatterplots showing induction of apoptosis with depletion of TTK. PANC-1 cells were transfected with control or a TTK targeted siRNA pool. 72 h post transfection cells were harvested and stained with the apoptotic marker Annexin V and the counterstained with propidium iodide to visualize necrotic cells. (G) quantitation of the apoptotic induction of cells used in F.