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. 2012 Mar 15;8(3):e1002583. doi: 10.1371/journal.pgen.1002583

Figure 5. The Fkh proteins and the APC work together to promote extended CLS and stress response.

Figure 5

(A) The FKH genes were deleted in apc5CA cells by multiple rounds of genetic crosses. The strains shown were grown overnight at 30°C in YPD, then spot diluted onto YPD plates and incubated at 30 or 37°C. (B) WT and apc5CA cells were transformed with plasmids expressing FKH1 or FKH2 from a galactose inducible promoter, or an empty vector control plasmid. Individual transformed colonies were grown overnight and then spot diluted onto SD ura- plates supplemented with either 2% glucose or 2% galactose. The plates were incubated at 30 or 37°C. (C) The mutants used above were grown to stationary phase and maintained in depleted media (DM) for the remainder of the experiment. Colony forming units were determined every other day and a survival curve was plotted. Standard error is shown for at least 3 repeats. (D) CLS was determined for the strains used above. Rather than maintenance in DM, the cells were washed once they reached stationary phase and maintained in H2O for the remainder of the experiment. Standard error is shown for at least 3 repeats. The experiments in (C) and (D) were started from the same cultures. (E) The panel of strains shown were grown overnight at 30°C to early log phase growth. Proteins were extracted and analyzed by Westerns to assess total histone H2B, H3 and H4 protein levels. A Ponceau S stained gel is included to shown equivalence of protein load. (F) WT, apc5CA and fkh1Δ fkh2Δ cells encoding BAR1 were grown to early log phase, then arrested with 2 µg/ml α factor for 1.5 hours in pH 3.5 YPD media. Another 2 µg/ml α factor was added followed by another hour of incubation. Cycloheximide was then added with continued incubation. Samples were removed at the times shown and Clb2 protein levels were determined by Western blotting. Antibodies against GAPDH were included as a load control. The same blot was divided and used for the Clb2 and GAPDH Westerns. Samples were taken before and after G1 arrest for FACS analysis.