Cells were synchronized in the G1 stage of the cell cycle by incubating exponentially growing cells in YPD medium at a concentration of 1x107 cells/ml with 4 μg/ml α-factor (GenScript) during 45 min, followed by addition of the same amount of α-factor and an additional 45 min incubation. Cells were released from the G1 arrest by filtration and extensive washing with prewarmed (30°C) YPD medium, and resuspension in fresh medium at the original cell concentration. Samples were taken at different times and flow cytometry was performed following standard procedures. The percentage of budded cells was determined under the microscope in parallel fixed samples.
α factor
Manufactured by GenScript
Sourced in United States
α-factor is a lab equipment product offered by GenScript. It is a pheromone that acts as a cell signaling molecule, triggering a response in yeast cells during mating. The core function of α-factor is to induce cell cycle arrest and prepare cells for mating.
Automatically generated - may contain errors
Lab products found in correlation
Nocodazole, by Merck Group (3 mentions)
α factor, by Merck Group (2 mentions)
Lsm 510 meta, by Zeiss (2 mentions)
Subtilisin, by Merck Group (1 mentions)
Poly di dc, by Merck Group (1 mentions)
T4 polynucleotide kinase, by Enzynomics (1 mentions)
Pcr primers, by Cosmogenetech (1 mentions)
Restriction enzymes, by Enzynomics (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
γ 32p atp, by PerkinElmer (1 mentions)
Methionine, by Merck Group (1 mentions)
Auxin, by Merck Group (1 mentions)
Rp01002, by GenScript (1 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
Hydrogen peroxide, by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Hydroxyurea hu h 8627, by Merck Group (1 mentions)
Axioskop 2 microscope, by Zeiss (1 mentions)
Cellquest software, by BD (1 mentions)
Benomyl, by Merck Group (1 mentions)
14 protocols using α factor
1
Cell Cycle Synchronization by α-Factor
2
Preparation of Radiolabeled Substrates and ssDNA
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Oligonucleotides used for the preparation of radiolabeled substrates and ssDNA competitor were synthesized and gel‐purified by Bioneer (Daejeon, Korea). [γ‐32P]ATP (3000 Ci·mmol−1) was purchased from Perkin Elmer (Waltham, MA, USA). PCR primers were synthesized by Cosmo Genetech (Seoul, Korea). Subtilisin, MMS, CPT, propidium iodide, and poly(dI‐dC) were purchased from Sigma‐Aldrich (St. Louis, MO, USA). M13mp18 sscDNA was acquired from New England Biolabs (Ipswich, MA, USA). α‐factor was purchased from GenScript (Piscataway, NJ, USA). Restriction enzymes, T4 polynucleotide kinase, and protein size markers were obtained from Enzynomics (Daejeon, Korea).
3
Cell Cycle Synchronization Protocols
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Cell cycle arrest and synchronization experiments were performed as previously described (Amon, 2002 (link)). Yeast cells were grown in yeast extract peptone (YEP) media unless differently specified in the figure legend. Carbon sources (glucose, raffinose, or galactose) were used at a final concentration of 2%. In all experiments cells were pre-arrested in G1 phase with 5 µg/ml α-factor (RP01002; Genscript) and released in a synchronous cell cycle or into the arrest of interest. To release cells from G1, cells were washed with 10 volumes of fresh medium and transferred into medium lacking the pheromone. When pertinent, drug(s) were added and restrictive conditions applied.
In detail: Temperature-sensitive alleles were inactivated by incubating the culture at the restrictive temperature of 37°C. cdc5-as1 and cdc15-as1 alleles were inhibited by adding to the media 5 µM of CMK (custom-made, Accendatech; Snead et al., 2007 (link)) or of 1NM-PP1 analogue 9 (A603003; Toronto Research Chemicals), respectively. Cells carrying an AID degron were inactivated with Auxin (I5148; Sigma-Aldrich) at a final concentration of 500 µM. Methionine (M9625; Sigma-Aldrich) was added at a final concentration of 8 mg/ml to inactivate the MET-CDC20 construct. HU (H8627; Sigma-Aldrich) was used at a final concentration of 10 mg/ml to arrest cells in S-phase.
In detail: Temperature-sensitive alleles were inactivated by incubating the culture at the restrictive temperature of 37°C. cdc5-as1 and cdc15-as1 alleles were inhibited by adding to the media 5 µM of CMK (custom-made, Accendatech; Snead et al., 2007 (link)) or of 1NM-PP1 analogue 9 (A603003; Toronto Research Chemicals), respectively. Cells carrying an AID degron were inactivated with Auxin (I5148; Sigma-Aldrich) at a final concentration of 500 µM. Methionine (M9625; Sigma-Aldrich) was added at a final concentration of 8 mg/ml to inactivate the MET-CDC20 construct. HU (H8627; Sigma-Aldrich) was used at a final concentration of 10 mg/ml to arrest cells in S-phase.
4
Yeast Strains and Plasmids Characterization
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All yeast strains and plasmids used in this work are listed in Tables S1 andS2, † respectively. The yeast strains were derived from BY4741 (ref. 31) and W303. Unless indicated otherwise, all fusion proteins were expressed from their endogenous promoter at the endogenous genomic locus, generated by replacing the endogenous gene using homologous recombination. α-Factor was obtained from Genscript (Luzern, Switzerland). Hydrogen peroxide, sodium chloride, sodium dodecyl sulfate, and vanadium oxide (vanadate) were purchased from Sigma Aldrich (Buchs, Switzerland).
Yeast strains for all experiments were grown in SC (synthetic complete) based media (0.17% yeast nitrogen base, 2% glucose, 0.5% NH 4 -sulfate and amino acids). Single yeast colonies were grown overnight, diluted 100-fold in fresh media and incubated at 30 °C for 4 h prior to loading into the chips.
Yeast strains for all experiments were grown in SC (synthetic complete) based media (0.17% yeast nitrogen base, 2% glucose, 0.5% NH 4 -sulfate and amino acids). Single yeast colonies were grown overnight, diluted 100-fold in fresh media and incubated at 30 °C for 4 h prior to loading into the chips.
5
Cell Cycle Arrest and Release
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Cell-cycle arrest at the end of G1 was induced in liquid YPD cultures (7.5 × 106 cells/ml) with 2 µg/ml α-factor (GenScript) for 2 h at 30°C. Cells were released from G1-arrest by the addition of 50 µg/ml Pronase. G1-arrest and release were validated by FACS.
6
Cell Cycle Arrest Induction Protocol
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Cells were grown in YPD to logarithmic phase at 30ºC, treated for 2 h with 3 μM α-factor (RP01002; GenScript, Piscataway, NJ) to arrest cells in G1, 0.2 M hydroxyurea (HU; H8627; Sigma-Aldrich, St. Louis, MO) to arrest cells in S phase, and 20 μg/ml nocodazole (M1404; Sigma-Aldrich) to arrest cells in G2/M. Samples were analyzed by FACS to confirm the cell cycle arrest using a Becton-Dickinson FACSort flow cytometer and Cell Quest software (BD Biosciences, Boston, MA). Cell cycle stages were determined based on nuclear position and cell morphology in propidium iodide–stained cells using the Zeiss Axioskop 2 microscope (Carl Zeiss, Peabody, MA) as described previously (Calvert and Lannigan, 2010 ).
7
Cell Cycle Synchronization Assay
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WT and mutant Orc4-IHΔ yeast strains were cultured in YPG medium before shifting to YPD medium to deplete the endogenously expressed Orc4 proteins for at least 18 h at 30 °C until OD600 of ~0.01 starter culture reached ~2. Strains were grown at 30 °C in YPD medium for all experiments. Cells were arrested at G1 with α factor (GenScript) at a concentration of 10 μg/ml for ~3 h and replenished every 60 min. G2/M arrests were achieved by culturing cells in medium containing 20 μg/ml benomyl (Sigma) for ~3 h. FACS analysis was performed, as previously described43 (link).
8
Quantitative 3D Yeast Cell Imaging
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All microscopy measurements were performed as described in (19 (link)) with slight modifications. Briefly, yeast cultures were grown to OD600 nm = 0.1–0.3 in synthetic complete (SC) medium containing 4% glucose at 30°C and synchronized in G1 phase with 10 μg/ml α-factor (GenScript) for 2 hours. Cells were then immobilized in microscopy chambers (Ibidi) coated with 2 mg/ml Concanavalin A (Sigma Aldrich) and washed thoroughly with warm medium to remove α-factor before microscope observation. Cells were imaged on a Marianas spinning-disk confocal microscopy platform (3i), using an Evolve EM-CCD camera (Photometrics). 488 and 561 nm lasers were used for excitation of GFP and tdTomato, respectively. Imaging was performed at 1 min intervals and at a temperature of 28°C for 3–4 h, using an ×63 oil objective (NA = 1.4) in 3D (12 z-sections, 0.7 μm apart). Time-lapse data were collected with SlideBook (3i) and exported to Matlab for analysis using a custom-made package (‘DotQuant’) that identifies, tracks and quantifies the fluorescent foci in each cell (19 (link)). Statistical analysis of the results was performed using Monte Carlo resampling with 1 000 000 iterations.
9
G1 Arrest and Release in Yeast
10
G1 Arrest and Release in Yeast
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S. cerevisiae cells were synchronized by G1-arrest using a 3-hour incubation in the presence of 10 μg/ml α-factor (GenScript). For release from G1-arrest, cells were washed with water and resuspended in YPD medium containing 15 μg/ml nocodazole (Sigma-Aldrich) to prevent cells from entering a second G1 and S phase.
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