Fig 1: Loss of AURKB activity in ESCs results in the formation of MTS. (A) Examples of MTS (obtained with APH treatment) shown. (B) Representative metaphase images of untreated control mouse ES129.1 cells (i) and those treated with either 0.2 µM APH (ii) or 1 µM AURKB inhibitor ZM447439 (iii) for 24 h. TEL-FISH analyses indicated that 24 h of 1 µM ZM447439 treatment resulted in an increase in MTS formation from an average of 2.3 of MTS/metaphase in untreated control cells to 8.5 MTS/metaphase in ZM447439 treated cells (P < 0.0001; N = 1000 chromosomes from three biological replicates), compared to an average of 7.3 MTS/metaphase in cells treated with 0.2 µM APH (P < 0.0001; N = 1000 chromosomes from three biological replicates) (iv and v). (C) Western blot analyses of AURKB and actin in ES129.1 cells subjected to scramble control siRNA and siRNA depletion of TERF1, TERF2 and AURKB, respectively (i). Representative images of metaphase ES129.1 cells subjected to scramble control siRNA (ii; negative control), 72 h of AURKB (iii) and TERF1 siRNA depletion (iv), respectively. About 72 h of AURKB depletion resulted in aberrant MTS formation, increasing from an average of 2.3 MTS/metaphase in cells subjected to scramble control siRNA depletion to 5.1 MTS/metaphase in AURKB-depleted cells (P = 0.0006, N = 1200 chromosomes from three biological replicates) (v and vi). As a comparison, 72 h of TERF1 siRNA depletion caused an average of 19.95 MTS/metaphase (P < 0.0001; Cv and vi). Magnified images of the boxed chromosomes in B and C are shown in the inset, with examples of MTS indicated by the arrowheads. Each point in scatterplots (Biv and Cv) represents of the number of MTS in a single metaphase spread, with error bars showing Q1, Q2 and Q3 values. P-values are indicated in column graphs (Biv and Cv). Scalebars represent 5 µm.