Increased <i>FKH1</i> or <i>FKH2</i> expression improves stress resistance and extends CLS and RLS.

<p>(A) Schematic representation of scheme used to integrate the <i>GAL1</i>/<i>10</i> promoter upstream of <i>FKH1</i> and <i>FKH2</i>. The <i>LEU2</i> PCR product, containing 300 basepairs of <i>LEU2</i> promoter (incorporated for selection purposes), was flanked by 60 basepairs of homology to the <i>FKH</i> promoter and to the <i>GAL1</i>/<i>10</i> promoter. The <i>GAL1</i>/<i>10</i> promoter PCR fragment was flanked by 60 basepairs of homology to the 3′ end of <i>LEU2</i> and to the 5′ end of the <i>FKH</i> gene. Cells were cotransformed with both products and selected on leu⁻ plates. Cells harboring <i>FKH1</i> and <i>FKH2</i> under the control of the <i>GAL1</i>/<i>10</i> promoter were generated by crossing the single integrated strains. (B) Cells overexpressing (OE) <i>FKH1</i> and/or <i>FKH2</i> from the <i>GAL1</i>/<i>10</i> 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) <i>FKH1-TAP</i> 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 <i>FKH1-TAP</i> and <i>FKH1</i> under their own promoter were used as controls. (D) <i>FKH2-TAP</i> OE cells were analyzed as described above for <i>FKH1-TAP</i> OE cells.</p

<i>Saccharomyces cerevisiae</i> strains used in this study.

<p><i>Saccharomyces cerevisiae</i> strains used in this study.</p

Model depicting how the APC and the Fkh proteins may interact under normal and stress conditions.

<p>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.</p

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

<p>(A) The <i>FKH</i> genes were deleted in <i>apc5^CA</i> 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 <i>apc5^CA</i> cells were transformed with plasmids expressing <i>FKH1</i> or <i>FKH2</i> 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, <i>apc5^CA</i> and <i>fkh1Δ fkh2Δ</i> cells encoding <i>BAR1</i> 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.</p

The APC and the Fkh proteins provide overlapping function to respond to oxidative stress.

<p>(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.</p

<i>FKH1</i> and <i>FKH2</i> encode redundant determinants of lifespan and stress response.

<p>(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 <i>fkh1Δ fkh2Δ</i> 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 <i>fkh1Δ fkh2Δ</i> 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.</p

Increased expression of the <i>FKH</i> genes increases lifespan and stress response.

<p>(A) The <i>FKH</i> 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.</p

The Fkh proteins are present in the nucleus of stationary phase cells.

<p>(A) Cells expressing endogenously TAP-tagged <i>FKH1</i> 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 <i>FKH1</i>- or <i>FKH2</i>-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.</p

DOX resistant MCF7 breast cancer cells are associated with chromatin alterations and DNA damage.

<p>(<b>A</b>) MCF7 cells before and after DOX selection were stained with DAPI to visualize DNA (blue) and with antibodies against BCRP (red). (<b>B</b>) Protein lysates were prepared from parental and selected MCF7 cells and analyzed by Western analyses using the antibodies shown. (<b>C</b>) Volcano plot of genes differentially expressed following the full DOX selection protocol compared to starting parental cells. The X-axis denotes expression changes with positive to the right and negative to the left. The Y-axis shows statistical significance (p-value) of the changes observed. The vertical red lines define the threshold for 2-fold positive and negative changes. (<b>D</b>) Volcano plot of differentially expressed genes following acute exposure to 1 µM DOX for 48 hours. The dots in green and yellow define the genes that were up-regulated and down-regulated, respectively. (<b>E</b>) Volcano plot demonstrating the differential expression of genes following the 2-week chronic exposure phase after the 48-hour treatment. The Tissue Factor Pathway Inhibitor family members are shown (TFPI1α, TFPI1β and TFPI2).</p

TFPI1 is expressed early in the drug selection process, but is not required for maintenance of the MDR state.

<p>(<b>A</b>) Parental and DOX selected MCF7 cells were treated with scrambled (S) or TFPI1 siRNA (+). A Western analysis using antibodies against TFPI1 show that silencing of TFPI1 was effective. eGFP was transfected along with the siRNA constructs and show that transfection efficiency was consistent. αTubulin was used as a load control. (<b>B</b>) Following 24 hours of siRNA treatment in DOX^Sel cells, 1 µM DOX was added for 48 hours. MTT was performed to determine cell killing. (<b>C</b>) Parental MCF7 cells were treated with 1 µM DOX for 48 hours, then maintained in 100 nM DOX for an additional 3 days. Protein samples were prepared every 24 hours and analyzed by Western blotting with the antibodies shown. (<b>D</b>) Parental MCF7 cells were incubated in 100 nM DOX for 5 days with samples removed every 24 hours for Western blotting with the antibodies indicated.</p