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Results: 7

1.
Figure 7

Figure 7. Model depicting how the APC and the Fkh proteins may interact under normal and stress conditions.. From: The Yeast Forkhead Transcription Factors Fkh1 and Fkh2 Regulate Lifespan and Stress Response Together with the Anaphase-Promoting Complex.

Under normal conditions, the Fkh proteins play a role in activating the APC. This in turn mediates progression through mitosis and maintenance of G1. This interaction is depicted by bold arrows. Under stress conditions, both the APC and the Fkh proteins respond via different mechanisms. The Fkh proteins likely promote the expression of stress response proteins. The APC acts according to a mechanism that remains uncharacterized, but likely requires ubiquitin-dependent processes.

Spike D. L. Postnikoff, et al. PLoS Genet. 2012 March;8(3):e1002583.
2.
Figure 2

Figure 2. The Fkh proteins are present in the nucleus of stationary phase cells.. From: The Yeast Forkhead Transcription Factors Fkh1 and Fkh2 Regulate Lifespan and Stress Response Together with the Anaphase-Promoting Complex.

(A) Cells expressing endogenously TAP-tagged FKH1 were grown to stationary phase and either left in DM or transferred to H2O for the remainder of the experiment. Samples were removed on days 1 and 5 for Western analysis using antibodies against the TAP epitope, or GAPDH as a load control. Fkh2-TAP was also observed in stationary phase cells (data not shown). (B) Cells expressing endogenously tagged FKH1- or FKH2-GFP were grown to day 5 stationary phase while maintained in DM. Cells were observed to harbor both Fkh1 and Fkh2 nuclear staining. (C) Day 13 stationary phase cells expressing Fkh2-GFP were imaged, showing reduced nuclear staining in cells maintained in DM. (D) The percentage of nuclear localized Fkh1-GFP or Fkh2-GFP was determined as cells aged in either DM or H2O.

Spike D. L. Postnikoff, et al. PLoS Genet. 2012 March;8(3):e1002583.
3.
Figure 6

Figure 6. The APC and the Fkh proteins provide overlapping function to respond to oxidative stress.. From: The Yeast Forkhead Transcription Factors Fkh1 and Fkh2 Regulate Lifespan and Stress Response Together with the Anaphase-Promoting Complex.

(A) CLS was performed using the strains shown in the presence of 25 mM H2O2. The cells were grown to stationary phase, and then H2O2 was added to the cultures. Colony forming units were determined every other day for the remainder of the experiment. Survival curves are shown. (B) The strains described above were used to determine resistance to oxidative stress. Day 5 stationary phase cells were exposed to 100 mM H2O2 for 1 hour at 30°C. Controls were not treated with H2O2. Diluted cells were then plated onto YPD plates until colonies formed. The percent survival was determined, with standard error shown for at least 3 replicates.

Spike D. L. Postnikoff, et al. PLoS Genet. 2012 March;8(3):e1002583.
4.
Figure 4

Figure 4. Increased expression of the FKH genes increases lifespan and stress response.. From: The Yeast Forkhead Transcription Factors Fkh1 and Fkh2 Regulate Lifespan and Stress Response Together with the Anaphase-Promoting Complex.

(A) The FKH OE cells were grown to stationary phase, then either maintained in DM, or 0.05% galactose was added. The cells were incubated for an additional 5 days, then split, with one half treated with 100 mM H2O2 for 1 hour. The other half served as the untreated control. Following the 1 hour incubation, the cells were diluted and plated onto YPD until colony forming units formed. Survival was determined by dividing the treated cells by the untreated cells. Standard error is shown for at least 3 replicates. (B) CLS was determined for the OE strains when maintained solely in DM (left panel) or in DM supplemented with 0.05% galactose (right panel). Standard error is shown for at least 3 replicates. (C) RLS was determined for the OE strains on 2% sucrose plates or sucrose plates supplemented with 0.1% galactose. Typical results are shown.

Spike D. L. Postnikoff, et al. PLoS Genet. 2012 March;8(3):e1002583.
5.
Figure 3

Figure 3. Increased FKH1 or FKH2 expression improves stress resistance and extends CLS and RLS.. From: The Yeast Forkhead Transcription Factors Fkh1 and Fkh2 Regulate Lifespan and Stress Response Together with the Anaphase-Promoting Complex.

(A) Schematic representation of scheme used to integrate the GAL1/10 promoter upstream of FKH1 and FKH2. The LEU2 PCR product, containing 300 basepairs of LEU2 promoter (incorporated for selection purposes), was flanked by 60 basepairs of homology to the FKH promoter and to the GAL1/10 promoter. The GAL1/10 promoter PCR fragment was flanked by 60 basepairs of homology to the 3′ end of LEU2 and to the 5′ end of the FKH gene. Cells were cotransformed with both products and selected on leu plates. Cells harboring FKH1 and FKH2 under the control of the GAL1/10 promoter were generated by crossing the single integrated strains. (B) Cells overexpressing (OE) FKH1 and/or FKH2 from the GAL1/10 promoter were grown overnight in 2% YPD, then spot diluted onto the plates shown. The plates were incubated at 30°C for 2 to 5 days. (C) FKH1-TAP OE cells were grown overnight in 2% glucose. The next day, the cells were washed and resuspended in media containing 2% galactose. Samples were taken every two hours for 10 hours for Western analysis using antibodies against TAP and GAPDH. Cells expressing FKH1-TAP and FKH1 under their own promoter were used as controls. (D) FKH2-TAP OE cells were analyzed as described above for FKH1-TAP OE cells.

Spike D. L. Postnikoff, et al. PLoS Genet. 2012 March;8(3):e1002583.
6.
Figure 1

Figure 1. FKH1 and FKH2 encode redundant determinants of lifespan and stress response.. From: The Yeast Forkhead Transcription Factors Fkh1 and Fkh2 Regulate Lifespan and Stress Response Together with the Anaphase-Promoting Complex.

(A) and (B) The cells shown were grown in CM (2% glucose) to stationary phase. The cells were either left in (A) depleted CM (DM), or (B) washed and resuspended in H20 for the remainder of the experiment. Colony counts were performed every other day throughout the experiment. The day when the colony count peaked was considered Day 1. Standard error is shown for at least 3 replicates. (C) Acute oxidative stress in stationary phase cells. WT and fkh1Δ fkh2Δ cells were grown to day 5 of stationary phase with maintenance in either DM or H2O. 100 mM H2O2 was then added to one half of each sample and incubated for 60 minutes at 30°C. Diluted cells were then plated on YPD media and the colony forming units were counted. Survival was determined by dividing the colony forming units following H2O2 treated by untreated samples. Standard error of at least 3 replicates is shown. (D) Chronic oxidative stress in mitotically active and stationary phase cells. WT and fkh1Δ fkh2Δ cells from overnight log phase cultures or day 5 stationary phase cultures were treated with 100 mM H2O2 at 30° for 1 hour, as above, then spot diluted onto YPD plates in the absence of stress. The plates were grown at 30°C for 3 days.

Spike D. L. Postnikoff, et al. PLoS Genet. 2012 March;8(3):e1002583.
7.
Figure 5

Figure 5. The Fkh proteins and the APC work together to promote extended CLS and stress response.. From: The Yeast Forkhead Transcription Factors Fkh1 and Fkh2 Regulate Lifespan and Stress Response Together with the Anaphase-Promoting Complex.

(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.

Spike D. L. Postnikoff, et al. PLoS Genet. 2012 March;8(3):e1002583.

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