Isoginkgetin

U2OS, HeLa and HeLa Luc/Luc-I cells were cultured in low glucose Dulbecco's modified Eagle medium (HyClone, Thermo Scientific, Stockholm, Sweden) supplemented with 10% fetal bovine serum (Gibco, Thermo Scientific, Stockholm, Sweden) and 2.5 μg/ml plasmocin (InvivoGen, Toulouse, France) at 37 °C in a humidified incubator under 5% CO₂. Direct repeated-GFP U2OS cells were cultured in high glucose Dulbecco's modified Eagle medium (HyClone) supplemented with 10% fetal bovine serum and 2.5 μg/ml plasmocin at 37 °C in a humidified incubator under 5% CO₂. Human GM00730 fibroblasts (Coriell Institute, Camden, NJ, USA) were cultured in minimum essential medium supplemented with 15% fetal bovine serum and 2.5 μg/ml plasmocin at 37 °C in a humidified incubator under 5% CO₂.
For treatment, 100 nM pladienolide B (Santa Cruz Biotechnologies, Heidelberg, Germany) or 50 μM isoginkgetin (Merck Millipore, Solna, Sweden) (final concentrations) were added to the culture medium 1–24 h prior to irradiation or harvesting, with the control cells receiving an equal volume of DMSO (Sigma-Aldrich, Stockholm, Sweden) as the volume of isoginkgetin used. Cycloheximide was added directly to the culture medium to give a final concentration of 50 μg/ml.

For some analyses, oocytes were treated with 100 µM Isoginkgetin (416154, Sigma Aldrich) alone, co-treated with 100 µM Isoginkgetin and 100 nM Reversine (R3904, Sigma Aldrich) or treated with vehicle solvent alone (DMSO) to assess the effects of interference with RNA splicing on meiotic maturation. In other experiments, oocytes were treated with 50 µM Triptolide (T3652, Sigma Aldrich) to gain insight into the role of transcriptional elongation during meiotic maturation. Control group oocytes were incubated in media containing the appropriate concentration of solvent (DMSO) only under similar conditions. All cultures were maintained at 37°C in MEM/BSA under 5% CO₂, 5% O₂ and N₂. Following culture, oocytes were immediately fixed for immunofluorescence analysis to assess the spindle microtubules, aMTOC components and chromosome configurations.