br We thank Veronica Rodriguez Bravo
We thank Veronica Rodriguez-Bravo and Sun Joo Lee for experimental assistance and Prasad Jallepalli and Bryan Tsou for helpful discussions. This work was supported by the Functional Genomics Initiative at Me-morial Sloan Kettering (E.A.F. and A.K.), NIH/NIGMS GM125996 (E.A.F), and NCI CA214812 to A.K. This work was prepared while E.A.F. was employed at Memorial Sloan Kettering Cancer Center. The opinions expressed in this article are the authorÕs own and do not reßect the view of the National Institutes of Health, the Department of Health and Human Services, or the US government.
Formal analysis and investigation, N.V.A., M.M.-O., and P.C.; Conceptualization, experiment design, and writing, N.V.A. and E.A.F.; Funding acquisition, E.A.F. and A. K.
DECLARATION OF INTERESTS
The authors declare no competing interests.
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A Cancer-Associated Missense Mutation in PP2A-Aa Increases Centrosome Clustering during Mitosis Noelle V. Antao, Marina Marcet-Ortega, Paolo Cifani, Alex Kentsis, and Emily A. Foley
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Figure S1. Analysis of PP2A activity and holoenzyme assembly in PP2A-AαP179R/+ cells, Related to Figure 2. (A-B) PP2A-Aα IPs in mitotic lysates from WT (+/+) or PP2A-AαP179R/+ (P179R/+). Okadaic acid was included in wash steps as indicated. The reaction was split and (A) associated proteins were analyzed by western blot or (B) incubated with a phosphopeptide substrate. Phosphate release was quantified using a molybdate dye-based spectrophotometric assay. Absorbance values were normalized to the maximum value per experiment. Mean + s.d. from three experiments is plotted. (C) Plotted is the normalized mean + s.e.m. of the amount of bait protein (PP2A-Aα) IP’d in the experiment in Figure 2D performed at least three times. (D) Western blot analysis of lysates (lanes 1-
Figure S2. Analysis of mitotic duration in PP2A-AαP179R/+ cells and centrosome amplification efficiency in Plk4-inducible cells, Related to Figure 3 and 4 (A) WT
(+/+) and PP2A-AαP179R/+ (P179R/+) cells were imaged live and the time from nuclear envelope breakdown to anaphase onset was measured. Clones a and b are independently derived cell lines. A box-and-whisker plot is shown. Whiskers indicate the 5-95 percentile range and circles indicate cells outside of this range. Result is representative of two experiments. (B) Maximum intensity projections of WT (+/+) and PP2A-AαP179R/+ (P179R/+) cells treated with cytochalasin D and then briefly with nocodazole. Cells were analyzed for microtubule re-growth by immunofluorescence upon nocodazole removal. (C-D) Cells were treated with cytochalasin D, fixed and analyzed by immunofluorescence. (C) Representative maximum intensity projection. (D) Multipolar anaphase incidence in cells with four centrin-1 foci is plotted (mean + s.e.m. of the experiment in (C) performed three times). (E-F) Tp53-/- WT (+/+) and PP2A-AαP179R/+ (P179R/+) cells with tet-inducible Plk4 expression were treated with dox or DMSO for 30 h, arrested in G2 with RO-3306 and dox or DMSO for 18 h, fixed and processed for