Extended Data Figure 8: BI 2536 treatment reduces PLK1 kinase activity in oocytes.

a, Schematic illustration of BI 2536 treatment in wild-type oocyte culture (left). Oocytes were treated with DMSO or BI 2536 (100 nM) during the indicated time periods, then washed and released into normal culture medium. The first polar body extrusion ratio (1st PBE) was counted at 10 h after GVBD (right). Error bars, mean ± s.e.m. from 3 independent experiments. The total number of oocytes used for each experiment is shown. b, Wild-type oocytes treated with DMSO or BI 2536 (during 6–7 h after GVBD) were fixed and immunostained for PLK1 substrate pCENP-U, ACA and DAPI at metaphase I (top). The relative pCENP-U intensity normalized to that of ACA is shown in the graph with mean + s.e.m. from 3 independent experiments. In each experiment, 10 kinetochores from an oocyte were quantified (n = 4 cells). **P < 0.01, unpaired t-test. c, Oocytes were treated with DMSO or BI 2536 during the period of 4–6 h after GVBD and stained for α-tubulin, CENP-C and DAPI (DNA) at the indicated stages in whole mount (related to Fig. 4d). Magnified images are shown to highlight the separation of sister kinetochores in BI 2536-treated oocyte in anaphase I. d, Chromosome spreads from control and BI 2536-treated wild-type oocytes at metaphase I (6 h after GVBD) and metaphase II (20 h after GVBD) were stained for REC8, CENP-C and DAPI (DNA) (related to Fig. 4d). Magnified images are shown to highlight the loss of cohesion in BI 2536 treated oocytes in metaphase II. e, Mlh1^−/− oocytes (C57BL/6 background) were treated with BI 2536 between 9–10 h after GVBD and stained for α-tubulin, CENP-C and DAPI (DNA) in whole mount (related to Fig. 4e). Magnified images are shown to highlight bi-oriented sister kinetochores at the spindle midzone. Scale bars, 5 μm (unless otherwise indicated).