In total, 150,000 cells were plated in 6-cm plates (for matched protein lysates and flow cytometry-based cell cycle analysis) or six-well plates (for matched LCMS-based metabolite measurements and flow cytometry-based cell cycle analysis) in DMEM with 10% FBS. The following day, cells were washed three times with PBS and medium was replaced with medium containing RO-3306 (Selleckchem), for which 4.5–9.0 μM RO-3306 was used, with the concentration for different lots of RO-3306 adjusted to obtain optimal synchronization for experiments. After treating cells for 18 h with RO-3306 (or DMSO for unsynchronized controls), cells were released from cell cycle arrest by washing three times with PBS and replacing the medium with untreated medium. Where relevant, medium containing either 50 nM AZ20 (or DMSO as vehicle) or guanine was added at the time of release from RO-3306. Cells were collected at the indicated timepoints after release from arrest. For each experiment, parallel samples for each timepoint were analysed by flow cytometry to assess cell cycle distribution.
Ro 3306
Manufactured by Selleck Chemicals
Sourced in United States, Japan
RO-3306 is a chemical compound used in laboratory research. It functions as a specific CDK1 inhibitor, which plays a key role in cell cycle regulation. The core function of RO-3306 is to provide researchers with a tool to study cell cycle dynamics and cell division processes.
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Lab products found in correlation
Thymidine, by Merck Group (9 mentions)
Nocodazole, by Merck Group (6 mentions)
Palbociclib, by Selleck Chemicals (5 mentions)
Ve 821, by Selleck Chemicals (5 mentions)
Thymidine, by Selleck Chemicals (4 mentions)
Mg132, by Selleck Chemicals (4 mentions)
Nocodazole, by Selleck Chemicals (4 mentions)
Mg132, by Merck Group (4 mentions)
Nu7441, by Selleck Chemicals (4 mentions)
Ku55933, by Selleck Chemicals (4 mentions)
Aphidicolin, by Merck Group (3 mentions)
Mln8237, by Selleck Chemicals (3 mentions)
Azd7762, by Selleck Chemicals (3 mentions)
Azd1775, by Selleck Chemicals (3 mentions)
5 ethynyl 2 deoxyuridine (edu), by Thermo Fisher Scientific (3 mentions)
Dimethyl sulfoxide (dmso), by Nacalai Tesque (3 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Volasertib, by Selleck Chemicals (2 mentions)
72 protocols using ro 3306
1
Cell Cycle Synchronization and Analysis
2
Synchronizing HeLa cells in anaphase
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HeLa cells were synchronized in anaphase by double thymidine release. Briefly, the cells were treated with 2 mM thymidine (T1895; Sigma-Aldrich) for 17 h, released for 8 h, followed by another round of thymidine treatment for 17 h. After 10.5 h of second thymidine release, the drug of choice was added onto the cells, as mentioned in the respective figure panels. To inactivate Aurora B kinase, cells were treated with 2 μM ZM447439 (S1103; Selleckchem). For initiating premature anaphase entry, cells were treated with 10 µM RO-3306 (S7747; Selleckchem) for 5 min. To inhibit Rock, cells were treated with 30 µM Y-27632 (Y0503; Sigma-Aldrich) for 1 or 12 h. To inhibit myosin II, cells were treated with 100 µM PNBB (Optopharma Ltd., DR-A-081) for 30 min or 12 h. Following drug treatments, the cells were fixed and immunostained.
For synchronization of cells in an anaphase-like state for immunofluorescence and immunoprecipitation analysis, HeLa Kyoto or HeLa Kyoto stably expressing AcGFP-Ect2r were synchronized in prometaphase with 100 nM Nocodazole for 17 h and released in 10 μM MG132 (S2619; Selleckchem) for 4 h to obtain metaphase-synchronized cell population. After that, cells were treated with 10 µM of Cdk1 inhibitor RO-3306 (S7747; Selleckchem) for 10 min to synchronize them in the anaphase-like state, as reported previously in Keshri et al. (2020) (link).
For synchronization of cells in an anaphase-like state for immunofluorescence and immunoprecipitation analysis, HeLa Kyoto or HeLa Kyoto stably expressing AcGFP-Ect2r were synchronized in prometaphase with 100 nM Nocodazole for 17 h and released in 10 μM MG132 (S2619; Selleckchem) for 4 h to obtain metaphase-synchronized cell population. After that, cells were treated with 10 µM of Cdk1 inhibitor RO-3306 (S7747; Selleckchem) for 10 min to synchronize them in the anaphase-like state, as reported previously in Keshri et al. (2020) (link).
3
Assessment of Cell Proliferation by CCK-8 Assay
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Cell proliferation ability was measured using the Cell Counting Kit-8 (CCK-8) following the manufacturer's instruction (Dojindo Molecular Technologies Inc, Dojindo, JPN). CDK1 inhibitor RO3306 (Selleck Chemicals, Houston, USA) was dissolved in DMSO at a concentration of 20 mM and added to the cell culture medium at final concentrations of 0, 2.5, 5, 10, 15 and 20 μM. HEC-1-B cells (1×104 cells/well) were cultured in a 96-well plate in culture medium with different concentration of RO3306 for 24, 48 or 72 h. Following culture, the medium was removed and 100 μl fresh medium containing Kit-8 reagents was added to each well for 2 h and optical density (OD) was detected at a wavelength of 490 nm using an enzyme-labeled analyzer. The optical density values were measured at least three times against reagent blank. Cell viability was determined by the formula: (Atreated/Acontrol) *100%.
4
Cell Cycle Synchronization and EdU Labeling
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BIR foci analysis was carried out as described with modifications34 (link). Cells were enriched in G2/M phase of cell cycle by sequential treatment with 2 mM Thymidine (Sigma #T9250) for 16 h, recovery in normal medium for 6 h and treatment with Cdk1 inhibitor RO-3306 (Sellekchem #S7747) for 12 h. During the final 1 h of RO-3306 treatment, medium was supplemented with 10 µM EdU. For flow cytometry, cells were fixed in 4% Formaldehyde/PBS for 15 min at room temperature. The cells were washed three times with PBS followed by permeabilization via gentle addition of pre-chilled 100% methanol and incubation overnight at −20 °C. The cells were washed once with PBS. Alexa488 was coupled to incorporated EdU by Click-iT chemistry using Click-iT Plus imaging kit (ThermoFisher #C10637) following the manufacturer’s protocol. Finally, cells were counterstained with DAPI at 0.2 µg/mL for 10 min. For cells grown on cover slips, the procedure outlined above was followed by incubation/washing cover slips in a 12-well plate followed by the IF-FISH procedure described earlier.
5
Visualizing Cell Cycle Regulation
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H2B-GFP-expressing control or KDM2A targeting sgRNA-transduced cells were seeded at 8 × 104 cells per well in fibronectin-coated 12-well 1.5 mm glass-bottom wells (Cellvis). Following culture in DMEM medium supplemented with 15% FBS for 24 h, cells were synchronized in the G2 phase by sequential treatment of thymidine and CDK1 inhibitor Ro-3306 (S7747, Selleckchem). Cells were firstly cultured in a medium containing 2 mM thymidine (Sigma-Aldrich) for 24 h. After washing twice with PBS followed by once with growth media, the cells were then released into fresh medium for 2 h before treatment with 10 mM CDK1 inhibitor for 16 h. Finally, cells were washed twice with PBS and once with growth media before being subjected to live cell imaging with Zeiss Cell Observer (ZEISS). Cells were monitored for 8 h at 5 min intervals. Movies are output by Zeiss ZEN software (ZEISS).
6
Pharmacological Inhibitors in Cell Cycle Regulation
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The pharmacological inhibitor thiostrepton was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Pharmacological inhibitors adavosertib, RO-3306, and volasertib (BI 6727) were purchased from Selleckchem (Houston, TX, USA). Pharmacological inhibitors aphidicolin and nocodazole were purchased from Cayman Chemical (Ann Arbor, MI, USA). Silencer Select siRNAs targeting FOXM1 (s5250), PLK1, Myt1 (s224087), Wee1 (s21), CDC25A (s2750), CDC25B (s2754), and CDC25C (s2758) and negative control, Lipofectamine RNAiMAX and Opti MEM were purchased from Thermo Fisher Scientific (Waltham, MA, USA). CDC2 siRNA (Cat # 3500S) was purchased from Cell Signaling Technologies (Danvers, MA, USA).
7
DNA Damage Response Pathway Evaluation
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Primary antibodies used for Western blot and immunofluorescence were as follows: mouse anti-γH2AX (05-636, Millipore, 1:1000 for immunofluorescence), mouse anti-53BP1 (612523, BD Transduction Laboratories, 1:500 for immunofluorescence), mouse anti-RPA23 (ab2175, Abcam, 1:1000 for Western blot ), rabbit anti-phospho-RPA (ab109394, Abcam, 1:50000 for Western blot ), and mouse anti-β-actin (E021020-01, Earthox, 1:1000 for Western blot). Chemical agents used were as follows: RO3306 (Selleck), CDK2 inhibitor II (Selleck), Aphidicolin (Sigma), camptothecin (Selleck), Cisplatin (Selleck), and Olaparib (Selleck).
8
Pharmacological Cell Cycle Arrest and Release
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For pharmacological inhibition of PLK1, BI2536 (Selleckchem), BI6727 (Volasertib, Selleckchem), and poloxin (Selleckchem) were added to cells at the indicated final concentrations in growth medium. For inhibition of Aurora A kinase, Alisertib (MLN8237, Selleckchem) was added to cells at the indicated final concentrations in growth medium. Arrest of cells in S-phase and mitosis was achieved by supplementing growth medium with 3 µM aphidicolin (Sigma) or 330 nM nocodazole (Sigma), respectively, for 16 h. Arrest of cells at the G2/M boundary was achieved by growth medium with 9 µM RO-3306 (Selleckchem) for 20 h. Release from cell cycle arrest was performed by three washes with pre-heated (37 °C) growth medium.
9
Mitotic Spindle Inhibitors Screening
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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.
10
Live-cell Imaging of Mitotic HeLa Cells
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Transfected mCherry-H2B HeLa cells were seeded onto Petri dishes with a 15-mm glass base (NEST), grown in DMEM media supplemented with 10% FBS, and treated with RO3306 (Selleck) to arrest in the G2 phase. Fluorescence time-lapse images were taken every 5 min for 24 h at 37 °C in 5% CO2 by using a laser-scanning confocal microscope (LSM880, Zeiss), with a 10×, 1.4-NA objective. Adobe Photoshop CC2018 was used for image processing.
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