Supplementary Figure 5: Expression of Wapl binding–deficient SA2 mutants bypasses Sgo1 requirement in cohesion protection and rescues cohesion fatigue.
From: Structure of cohesin subcomplex pinpoints direct shugoshin-Wapl antagonism in centromeric cohesion
(a) Anti-SA2, anti-Myc, and anti-tubulin blots of lysates of HeLa cells transfected with the indicated siRNA and plasmids. WT, wild type. (b) Four major types (I-IV) of metaphase spreads of cells in a, stained with DAPI (blue) and the kinetochore marker CREST (red). Selected sister chromatids were magnified and shown in insets. Scale bar, 5 μm. (c) Quantification of the percentage of cells in a with type III and IV chromosome morphologies as in b. Error bars, range (n = 2, independent experiments). The K330E mutant was tested only once. (d) Quantification of the percentages of mitotic HeLa cells (transfected with the indicated Myc-SA2 plasmids, arrested in nocodazole, and released into medium containing MG132 for 2 hrs) that had types III/IV chromosome morphology. WT, wild type. Error bars, s.d. for mock, WT, D326K (n = 3, independent experiments); range for K330E and Y331A (n = 2, independent experiments).
