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. 2018 Aug 6;7:e36898. doi: 10.7554/eLife.36898

Figure 7. Mitotic progression and cytokinesis after nocodazole washout does not require de novo transcription.

(A) Schematic representation of the experimental protocol used to study the effect of the transcription inhibitors ActD and triptolide on mitotic progression. (B) Selected time frames from phase contrast microscopy of HeLa cells treated after the NOC washout with DMSO, ActD or Triptolide. Images were acquired every 20 min. For control (DMSO) and Triptolide (Triptolide), arrows highlight a normal mitotic progression after nocodazole washout. For ActD-treated cells (ActD), arrows highlight a cell that fails to divide after release from nocodazole arrest. Scale bar = 10 µm. Time = hr:min. (C) Scattered plot showing the mitotic duration between nocodazole washout and anaphase onset in control (DMSO), ActD and Triptolide treated HeLa cells (DMSO, 79.1 ± 28.8 min, n = 116; ActD, 140.5 ± 55.7 hr, n = 79; Triptolide, 86.2 ± 31.9 hr, n = 113, median ±SD from a pool of two technical replicates, n.s. p>0.05, ****p≤0.0001, Mann-Whitney Rank Sum Test). (D) Cell fate of mitotic HeLa cells treated with the same drugs as in 7B.

Figure 7.

Figure 7—figure supplement 1. Actinomycin D promotes cytokinesis failure.

Figure 7—figure supplement 1.

(A) Representative immunofluorescence images of HeLa cells treated with DMSO, ActD or Triptolide for 2 hr after the NOC washout with the indicated antibodies. For ActD-treated cells, arrow highlights a cell with missegregated DNA in the spindle midzone. Scale bar = 5 µm. Dashed square encompasses a DNA bridge on the midzone and respective 5x magnification.