Mitotic Synchronization and Post-Mitotic Analysis of ES Cells

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.

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Chervova A., Molliex A., Baymaz H.I., Coux R.X., Papadopoulou T., Mueller F., Hercul E., Fournier D., Dubois A., Gaiani N., Beli P., Festuccia N, & Navarro P. (2024). Mitotic bookmarking redundancy by nuclear receptors in pluripotent cells. Nature Structural & Molecular Biology, 31(3), 513-522.

Publication 2024

RO-3306 SML0569 M1404 TRIzol

Corresponding Organization :

Other organizations : Université Paris Cité, Institut Pasteur, Centre National de la Recherche Scientifique, Epigénétique et Destin Cellulaire, Institute of Molecular Biology, La Ligue Contre le Cancer

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Variable analysis

independent variables

ES cell lines: E14Tg2a, EKOiE, EKOie–NrKO, FLAG–Nr5a2, NR5A2–GFP/ESRRB–mCherry, ESRRB/NR5A2–GFP, ESRRB/NR5A2–IAA
Depletion of ESRRB/NR5A2 with 0.5 mM auxin (5-Ph-IAA)

dependent variables

Gene expression changes during post-mitotic release (at 20, 30, 40, 50, 60, 90, and 120 minutes)

control variables

Serum and leukemia inhibitory factor culture conditions
Mitotic ES cells (>95% purity) obtained using CDK1 inhibitor RO-3306, nocodazole, and shake-off
Cells seeded in separate dishes (one per time point), uncoated with gelatin

controls

Positive control: Asynchronous cells treated with 0.5 mM auxin for 5 hours
Negative control: Not explicitly mentioned

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