Sml0569

ES cells—E14Tg2a, EKOiE^{5 (link)}, EKOie–NrKO^{14 (link)}, FLAG–Nr5a2 (ref. ^{14 (link)}), NR5A2–GFP/ESRRB–mCherry^{14 (link)}, ESRRB/NR5A2–GFP lines and ESRRB/NR5A2–IAA ESCs (see details below)—were cultured on serum and leukemia inhibitory factor conditions as previously described^{11 (link)}. Mitotic ES cells (>95% purity as assessed by 4′,6-diamidino-2-phenylindole staining and microscopy) were obtained using a double synchronization method based on the CDK1 inhibitor RO-3306 (10 μM; Sigma, SML0569), nocodazole (50 ng ml⁻¹; Sigma, M1404) and shake-off, as previously described^{9 (link)}. For post-mitosis analyses, cells were seeded in separate dishes (one per time point), purposely uncoated with gelatin and lysed in cold TRIzol (ThermoFisher, 15596026) 20, 30, 40, 50, 60, 90 and 120 min after release from the mitotic block^{9 (link)}. ESRRB/NR5A2 depletion was achieved with 0.5 mM auxin (5-Ph-IAA BioAcademia, 30-003), added during the 5 h of nocodazole block and maintained during the whole post-mitotic release. Asynchronous cells were treated in parallel during 5 h.

Human HeLa cervix carcinoma epithelial cells were cultured in DMEM supplemented with 10% fetal calf serum, 2% L-glutamine, and 2% penicillin/streptomycin in a humidified atmosphere at 37 °C in 5% CO₂. Preliminary comparisons of cell cycle synchronization protcols are described in detail in Supplementary Materials and Methods. For proteomic analysis, 5 × 10⁶ HeLa cells were grown in 150 cm² flasks in the presence of RO3306 (9 μM; SML0569, Sigma) for 20 hours, then released in RO3306-free medium, monitored under an inverted microscope and collected at round-up by mechanical shake-off. For automated image acquisition, cells were grown on 4-chamber Culture Slides (Falcon) (8 × 10⁴ cells per 1.7 cm² well), and either left to cycle asynchronously, or synchronized using RO3306 as above and fixed 60 minutes after RO3306 wash-out. Where indicated, cells were treated with 50 μM Importazole (Sigma) for the last 2–3 hours of culture before harvesting the cells.

The concentrations used for the MAPK pathway inhibitors (MEKi-1/2 and ERKi) were determined experimentally to be the doses at which phosphorylation of ERK1/2 and RSK1/2/3 returned to baseline despite the presence of oncogenic BRAF expression. The reagents used in these studies are as follows: MEKi-1: U0126 (Selleck Chemicals), 10 µM; MEKi-2: Trametinib (GSK1120212), 20 nM (Selleck Chemicals); ERKi: SCH772984, 20 nM (Selleck Chemicals); Hydroxyurea, 1 mM (Selleck Chemicals); doxycycline, 1 µg/mL (Sigma-Aldrich D9891); thymidine, 2.5 mM (Sigma-Aldrich T1895); RO-3306, 7 µM (Sigma-Aldrich SML0569)