The capacity of caffeine to advance cell cycle progression. Additionally they deliver additional evidence that caffeine can advance entry into mitosis but slows progression by means of cytokinesis. Concurrently, caffeine partly compensates for the lack of a spindle checkpoint by delaying progression through cytokinesis (Figs 2E and 3A ).2014 The Authors. Molecular Microbiology published by John Wiley Sons Ltd., Molecular Microbiology, 92, 777782 J. P. Alao et al.2014 The Authors. Molecular Microbiology published by John Wiley Sons Ltd., Molecular Microbiology, 92, 777Stabilized Cdc25 overrides checkpointsFig. 2. Caffeine modulates cell progression in S. pombe. A. Wt, rad3, and cds1 strains have been exposed to 10 mM caffeine. Cibacron Blue 3G-A Epigenetics Samples had been harvested in the indicated time points and fixed in 70 ethanol. Cells were stained with aniline blue plus the septation index determined by fluorescence microscopy. At least 200 cells had been counted for every time point. Error bars represent the mean of at least 3 independent experiments S.E. B. Wt, rad3, and cds1 strains were simultaneously exposed to 20 mM HU and 10 mM caffeine. Samples had been harvested at the indicated time points, fixed in 70 ethanol and analysed by FACS. Arrows indicate differential prices of cell cycle progression. C and D. wee1 mutants have been treated as within a and B. Arrows indicate differential prices of cell cycle progression. Wt septation index from A was included for clarity. E. nda3-KM311 mad2 mutants had been incubated at 18 untreated (Manage), treated with 50 g ml-1 MBC, 50 g ml-1 MBC and ten mM caffeine, or 10 mM caffeine for four h. Equal cell numbers had been spotted onto YES agar plates and incubated for three days. F. nda3-KM311 and nda3-KM311 mad2 mutants have been incubated at 18 inside the absence (top rated panel) or presence (bottom panel) of 10 mM caffeine. Samples had been harvested at the indicated time points and analysed by FACS. G. nda3-KM311 and nda3-KM311 mad2 were treated as in F. Samples have been harvested in the indicated time points and fixed in 70 ethanol. Cells were stained with aniline blue along with the septation index determined by fluorescence microscopy. No less than 200 cells were counted for each time point. Error bars represent the imply of no less than three independent experiments S.E.Caffeine advances cell cycle progression via Cdc25 The cdc2-3w allele abolishes the requirement for Cdc25mediated entry into mitosis and activation of the replication checkpoint. Mutants carrying the cdc2-3w allele remain below the handle of Wee1 phosphorylation and deletion of Cdc25 outcomes in enhanced cell length (Enoch et al., 1992; Basi and Enoch, 1996) (Fig. 4A). To further investigate the part of Cdc25 in mediating the effects of caffeine, we compared its impact on cell cycle progression in cdc2-3w and cdc2-3w cdc25 mutants. Exposure of cdc2-3w mutants to ten mM caffeine induced the sustained accumulation of septated cells using a 4C DNA content material. In contrast, caffeine exerted only minor effects on cell cycle progression in cdc2-3w cdc25 mutants (Fig. 4B and C). A simultaneous improve in each the number of binucleates and the septation index was observed (Supplementary Fig. S4B). It remains unclear if the slight improve in the 4C DNA peak reflects a delay within the cell cycle progression with the cdc2-3w cdc25 mutant. Exposure of cdc2-3w mutants to caffeine in the presence of latrunculin B (Lat B) (to be able to inhibit cytokinesis), demonstrated that progression via mitosis as well as the subsequent S phase was only moderatel.
