Extended Data Fig. 1: Human cells express alternative isoforms of Cdc20. Related to Fig. 1.
From: Alternative CDC20 translational isoforms tune mitotic arrest duration
(A) Western blot showing mitotic HeLa cells collected by shake-off after overnight treatment with 330 nM nocodazole. Lysates alone, with buffer only, or with buffer and lambda phosphatase treatment were probed using antibodies recognizing the C-terminus of human Cdc20 (aa 450–499). GAPDH was used as loading control. (B) Western blot showing multiple human cancer cell lines (A549, DLD-1, and U2OS) and the non-transformed human hTERT RPE-1 cells treated with control or Cdc20 siRNAs, as well as the mouse NIH/3T3 cell line. Cdc20 protein species were detected with antibodies recognizing the C-terminus of human Cdc20 (aa 450–499). β-actin or GAPDH was used as loading control. (C) Sequence information for the homozygous ∆M1 mutant cell line lacking the canonical M1 ATG start codon. The DNA sequence of the genomic locus was determined by next-generation sequencing. (D) Sequence information for the M1-stop mutant cell line showing insertions of 53 nt and 105 nt respectively after the L14 residue. Underlined are premature stop codons that are in-frame with the M1 ATG start codon for both CDC20 alleles. The DNA sequence of the genomic locus was determined by next-generation sequencing. (E) Growth curves of control HeLa compared to the ∆M1 and M1-stop mutant cell lines. (F) Graph showing mitotic duration for control HeLa compared to the ∆M1 and M1-stop mutant cell lines. Each point represents a single cell. The bars correspond to the mean. Indicated are the mean mitotic duration ± standard deviation across two experimental replicates, with replicates shaded-coded. The total number of cells analysed is indicated. Statistics from Student’s two-sample t-Test with two-tailed distribution (****p < 0.0001, NS not significant).
