Zm447439

The CENP-E inhibitor GSK-923295 (MedChemExpress LLC # HY-10299) was used at 200 nM. NOC (VWR #80058-500) was used at 100–500 ng/ml. STLC (Tocris #2191) was used at 25 μM. ProTAME (Concept Life Sciences custom synthesis) was used at 25 μM. Okadaic acid (LC Labs O-5857) was used at 200 nM. RO-3306 (Selleck #S7747) was used at 10 μM. AZ3146 (R&D Systems #3994/10) was used at 2μM. BI2536 (Synthesized in-house) was used at 100 nM. MLN8237 (Selleck #S1133) was used at 10–50 nM. ZM447439 (Tocris Bioscience #2458/10) was used at 2 μM. UMK57 (Aobious #AOB8668) was used at 100 nM.

All animal experiments were subjected to ethical review and done under the authorisation B-75-1308, according to current French guidelines. Adult CD-1 and C57BL/6 mice (control, Bub1KD, BubR1−/−, Mps1ΔN) were sacrificed, and ovaries dissected to obtain oocytes at the GV (Germinal Vesicle stage). Only oocytes undergoing GVBD up to 90 min after release were used. Pools of oocytes undergoing GVBD at the same time ( ± 15 min) were used for experiments. The Mps1 inhibitor Reversine (Cayman Chemical Research, 10004412) was added at GVBD at a final concentration of 0.5 μM. MG132 (Sigma-Aldrich, C2211) was added at GVBD + 3h at a final concentration of 20 μM and ZM447439 (Tocris, 2458) at GVBD at a final concentration of 10 μM. Oocytes were cultured in self-made M2 medium without CO₂. In vivo matured oocytes were obtained by injecting mice intraperitoneally with 5 UI of pregnant mare’s serum (PMS, Intervet) and 48 h later with 5 UI of human gonadotrophin (Intervet). Metaphase II oocytes surrounded by follicular cells were collected from the oviduct and incubated in M2 medium. Oocytes were separated from the follicular cells by adding hyaluronidase (Sigma-Aldrich, H4272). The zona pellucida was removed by exposing oocytes to low pH of tyrode acid solution^{29 (link)}, for both chromosome spread and whole-mount oocyte staining.