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

DOX selection induces HIF1α while hypoxia induces DOX resistance.

<p>(<b>A</b>) Protein lysates prepared from parental and DOX^Sel MCF7 cells were analyzed using HIF1α, PAR-1 and GAPDH antibodies. (<b>B</b>) Cells were exposed to 1% oxygen for 48 hours to induce hypoxia in the presence and absence of 1 µM DOX. Normoxic DOX treated cells (21% O₂) were used as controls. Survival was determined using an MTT assay. The experiment was performed twice with MTT assays done in triplicate. Standard error of the mean is shown. (<b>C</b>) A hypoxia time course was performed with protein lysates prepared at the times indicated. Westerns were performed using the antibodies shown.</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

Enhanced clearance of protein aggregates in yeast and worm <i>rer1</i> mutants.

<p><b>A.</b> Live cell imaging of α-syn-GFP after a 4 hr induction in 0.2% galactose. TM (0.02 μg/ml) was added to the cultures after an initial induction in galactose and cells were imaged 2 hours later. In a marked contrast to untreated cells, the GFP foci were largely vacuolar in <i>rer1Δ</i> or wild type cells treated with TM (arrows). <b>B.</b> GFP immunoblot in whole cell lysates prepared from cells in (<b>A)</b>. The intravacuolar proteolysis of α-syn-GFP generates the transiently stable GFP moiety detected as a discrete fragment in SDS-PAGE. <b>C.</b> Fluorescent images of transgenic hermaphrodites stably expressing α-syn-GFP from a body wall muscle promoter (<i>Punc-</i>_<i>54</i>::<i>α-syn</i>::<i>GFP</i>). Arrows denote a subset of the α-syn-GFP foci. Worms co-expressing heat shock protein torsinA (<i>tor-2</i>) in the same cells (<i>P</i>_{<i>unc-54</i>}::<i>tor-2</i>) served as a positive experimental control. The average number of α-syn-GFP foci per worm scored from digitized Z-stacked images ± s.e.m are plotted in <b>D</b> (n >20).</p

A genome scale screen for isolating mutants with extended mitotic lifespan in the yeast <i>S</i>. <i>cerevisiae</i>.

<p><b>A.</b> The screen work flow. The design rationale is detailed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005429#pgen.1005429.s001" target="_blank">S1 Fig</a>. <b>B.</b> Cy5:Cy3 signal ratios of mutants maintained in a dividing state for 16 days. Ranked values were log₂ normalized and projected. Of the starting collection of 3762, 52 mutants that maintained negative log₂ Cy5:Cy3 signal ratios at both day 6 and day 16 and displayed signal ratios <-2.3 at day 16 were classified as potentially long-lived (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005429#pgen.1005429.s003" target="_blank">S3 Fig</a>). <b>C.</b> Broad functional clustering of the putative longevity genes isolated in this screen using GO Ontology. Genes that function in protein modification and trafficking across the ER-Golgi network are outlined.</p

TFPI1 mRNA is elevated in human tumor samples when BCRP and MDR-1 are also elevated.

<p>1223 datasets from patients with breast (529), ovarian (539) or colon (155) tumors were gathered from an Agilent expression study. mRNA expression of BCRP was followed in these samples. Those with reduced and increased expression levels were segregated and pooled forming the sets “BCRP down” (1204) and “BCRP up” (19), respectively. In each pool, the expression of the shown genes was determined. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084611#s2" target="_blank">methods</a> for an explanation of the box plot.</p

Gene ontology list of differentially altered functions following selection of DOX resistant MCF7 cells.

<p>(<b>A</b>) Cellular functions involving up-regulated genes. (<b>B</b>) Cellular functions affected by the down-regulated genes. The number of genes in each category is shown.</p

TFPI1 overexpression increases DOX resistance and levels of procancer proteins, consistent with TFPI1 playing an early role in the MDR transition.

<p>(<b>A</b>) Parental MCF7 cells were transfected with an empty vector construct, or a construct overexpressing TFPI1. After 24 hours, the cells were harvested and prepared for protein analyses using the antibodies shown. (<b>B</b>) Cells were transfected with a TFPI1 expressing vector or the empty vector, and left for 24 hours. Next, the cells were treated with 1 µM DOX for an additional 24 hours. MTT was performed to determine cell killing. The MTT assay was done in triplicate with the standard error of the mean indicated. (<b>C</b>) The lysates used above were used to assess levels of the proteins shown. (<b>D</b>) A schematic representation of a possible model for how DOX exposure leads to DOX resistance. Increased TFPI1 protein could be p53-dependent (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084611#s4" target="_blank">Discussion</a>), while elevated HIF1α protein could be through HIF1α stabilization.</p