Extended Data Fig. 4: Truncated Cdc20 isoforms are inefficient targets of the SAC and promote mitotic slippage. Related to Fig. 2.
From: Alternative CDC20 translational isoforms tune mitotic arrest duration
(A) Cumulative frequency distribution showing the fraction of mitotic cells over time post-mitotic entry for HeLa, ∆M1, and M1-stop cells treated with 10 µM STLC alone or in combination with the APC/C-inhibitor proTAME (12 µM). The total number of cells included in the analyses are indicated in brackets. (B) Cumulative frequency distribution as in (A) for HeLa, ∆M1, and M1-stop cells treated with 10 µM STLC and 100nM of either control siRNAs or Cdh1 siRNAs. (C) Cumulative frequency distribution as in (A) for HeLa, ∆M1, and M1-stop cells treated with 10 µM STLC and either expressing endogenous Cdc20 protein and treated with control siRNAs or upon Cdc20 replacement with ectopic wild-type CDC20 cDNA. (D) Mitotic duration of individual HeLa cells expressing doxycycline-inducible Cas9 and sgRNAs recognizing different regions within the CDC20 gene. Unperturbed mitotic progression or mitotic arrest behaviour were monitored upon treatment with DMSO or 10 µM STLC respectively. Cells entering mitosis in the first 350–400 min of time lapse experiments were included in the analyses. Open red circles indicate cells that exit mitosis. Closed black circles indicate cells that remained arrested in mitosis till the end of the time lapse. Bars correspond to the median. (E) Representative immunofluorescence images of Bub1 or Mad2 localization to kinetochores immuno-stained with anti-centromere antibodies (ACA). Images are maximum intensity projections of deconvolved Z-stacks of selected mitotic cells from control HeLa or mutant ∆M1 or M1-stop cell lines treated with nocodazole. Images were scaled individually to highlight kinetochore localization. Scale bar, 5 µm. (F) Cumulative frequency distribution as in (A) except for HeLa, ∆M1, and M1-stop cells treated with 10 µM STLC alone or in combination with the Mps1-inhibitor AZ3146 (4 µM). (G) Cumulative frequency distribution as in (A) except for HeLa, ∆M1, and M1-stop cells treated with 10 µM STLC and either control siRNAs or Mad2 siRNAs.
