(A) Normalized expression of Mcl1 in asynchronous control HeLa cells or cells treated with ActD, triptolide or α-amanitin (ActD, 0.23 ± 0.02, Triptolide, 0.14 ± 0.03; α-amanitin, 1.13 ± 0.2, median ±SD from three technical replicates, n.s. p>0.05, ****p≤0.0001 relative to control, t test). (B) Scatter plot showing the duration of the mitotic arrest of control (NOC) and HeLa cells treated with Triptolide (NOC + Triptolide) after nocodazole treatment. The red line represents the mean and the error bars represent the standard deviation from a pool of at least two independent experiments. (NOC, 19.2 ± 7.0 hr, n = 386; NOC + Triptolide, 30.1 ± 14.7 hr, n = 301; median ±SD; n.s. p>0.05, Mann-Whitney Rank Sum Test) (C) Cell fate of mitotic HeLa cells treated with the same drugs as in 4B. (D) Representative immunofluorescence images of HeLa cells treated with DMSO (NOC) or Triptolide for 4 hr (NOC + Triptolide) after nocodazole treatment with the indicated antibodies. Scale bar = 5 µm. 10x magnification of a pair of kinetochores are shown in the right. Dashed circle encompasses a single kinetochore and the arrow indicates the position of centromeric Aurora B. Scale bar = 0.5 µm. (E) Normalized ratio of pAurora B/ACA fluorescence signal at inner centromere of NOC and NOC + Triptolide. Each dot represents an individual kinetochore. The red line represents the mean of all quantified kinetochores and the error bars represent the standard deviation from a pool of two independent experiments. (NOC, 1.00 ± 0.48, n = 474; NOC + Triptolide, 1.05 ± 0.00.40, n = 296; n.s. p>0.05 relative to control, Mann-Whitney Rank Sum Test) (F) Selected time frames from fluorescence microscopy of HeLa LAP-Aurora B cells treated with DMSO or Triptolide after nocodazole treatment. Images were acquired every 10 min. Scale bar = 5 µm. Time = h:min. (G) Relative fluorescence intensity curves of centromeric GFP-Aurora B for control and Triptolide-treated HeLa cells after nocodazole treatment. Fluorescence intensities were normalized to the level at time = 0. The curves depict mean GFP-Aurora B fluorescent intensity from analyzed cells (NOC n = 9; NOC + Triptolide n = 8; from time = 0 to 30 time frames), and error bars represent the standard deviation (n.s. p>0.05 relative to control, Analysis of covariance). Plus symbol on the graph indicates the time when the drug was added.