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To confirm the efficiency of synchronization, cells were fixed and analyzed by FACS at all time intervals.
For synchronization, cells were arrested at the G1/S boundary by incubation in the presence of 2.0 mM thymidine for 14 16 hrs twice with a 10 hr interval of growth without the drug.
We acquired S. cerevisiae cell-cycle data from the alpha factor synchronization study described in Pramila et al. 2006 [ 27] and S. bayanus alpha factor synchronization cell-cycle data described in [ 35] (available at GSE16544).
Following 24 h of synchronization, cells were treated with Pso IC50 value or DMSO (vehicle).
G0 - G1 synchronization cells were treated with 10-8 M of 17 β - estradiol (E2).
For synchronization, cells were treated with 200 ng/ml nocodazadole (Sigma).
To control for cell synchronization, cells for the + N and –N conditions were harvested at the same time of day.
To achieve phase synchronization, cells were arrested in the G0 phase (3) and released for different periods of time.
For pro-metaphase synchronization, cells were incubated for 18 h with DMEM medium containing 0.1 μg/ml of nocodazole (Fisher Scientific).
In the former method of synchronization, cells arrest at the point of cell cycle entry in late G1 known as START (Hartwell et al., 1974).
For synchronization, cells transfected with miR-NC or miR-188 were treated with 2 mM hydroxyurea (Sigma-Aldrich, H8627) for 16 h to block cell at G1 phase.
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