Validation of BaFiso assay using a panel of test compounds.

<p>(A) Analysis of the general toxicity of compound treatment. The total cell numbers in each well were determined by nuclear counterstain with the far-red fluorescent DNA probe DRAQ5. The number of DRAQ5-stained nuclei was determined after exposure to 30 µM Cisplatin (Cis), 100 µM Minerval (Min), 500 nM Akt Inhibitor X (AIX), 20 µM Genistein (Gen), 1 nM Leptomycin B (LMB), 20 nM Staurosporine (Stau), 1 µM UCN-01, 20 nM Raf1 Kinase Inhibitor (RKI), 20 µM LY294002 (LY), 10 µM Etoposide (Eto) and 1 mM lithium chloride (LiCl) for 12 hours and compared to vehicle treatment. (B) Equal numbers of BCS and BYA cells were co-cultured in IL-3-free medium. We exposed the paired BaFiso cell lines to 3 µM, 30 µM and 300 µM Cisplatin (Cis), 25 µM, 100 µM, 200 µM Minerval (Min), 50 nM, 500 nM, 5 µM Akt Inhibitor X (AIX), 200 nM, 20 µM, 50 µM Genistein (Gen), 0.5 nM, 1 nM, 4 nM Leptomycin B (LMB), 2 nM, 20 nM, 10 µM Staurosporine (Stau), 200 nM, 1 µM, 10 µM UCN-01; 5 nM, 20 nM, 200 nM Raf1 Kinase Inhibitor (RKI), 1 µM, 20 µM, 50 µM LY294002 (LY), 100 nM, 10 µM, 100 µM Etoposide (Eto) and 100 µM, 1 mM, 10 mM lithium chloride (LiCl), and Dimethyl sulfoxide (DMSO) as a negative control (striped bar). Three images specific for ECFP, EYFP or DRAQ5 from each well were acquired using BD Pathway Bioimager. The ECFP/EYFP ratio was determined by dividing the number of ECFP positive cells by the number of EYFP positive cells. Light, dark grey and black bars represent low, medium and high concentrations of the corresponding compounds, respectively. The data shown here represents three independent experiments. The average Z' value for BaFiso was 0.53. (C) Untreated, co-cultured BaFiso cells imaged before exposure to Akt Inhibitor X and (D) 12 h after treatment with 5 µM AIX.</p

The generation of BaFiso cell lines.

<p>Ba/F3 cells were transduced with retroviral supernatant carrying pBabePuro-EYFP or pBabePuro-ECFP. Cell clones were established and sorted in a fluorescence activated cell sorter (FACS) to generate lines homogeneously expressing the corresponding fluorescent tags. (A) and (C), viable Ba/F3 cells show robust and homogeneous expression of the respective fluorescent protein. (B) and (D), corresponding light field views. (E), Generation of stable BaFiso cell lines. Clonal Ba/F3 cells stably expressing EYFP (BY) or ECFP (BC) were used to generate stable BaFiso cell lines that co-express yellow fluorescence and myr-Akt (BYA), or cyan fluorescence and STAT5A1*6 (BCS). Cell clones were established and analyzed. (F), Analysis of transgene expression and downstream activation of the corresponding signaling pathways by western blotting. Antibodies against total Akt, Stat5a, Flag phospho-Akt (Ser473), phospho-p70 S6 (Thr389) and Pim-1 were used and the signals normalized to the respective α-tubulin levels.</p

Schematic overview of the BaFiso assay system.

<p>BaFiso consists of paired isogenic cell lines that have been engineered to acquire IL-3 autonomous growth through constitutive activation of Akt or Stat5 signaling. The two cell lines to be compared are individually tagged with either yellow or cyan fluorescent proteins. Equal numbers of yellow and cyan cells were co-cultured, treated with compounds and the change in the relative cell number was calculated on the basis of the distinct fluorescent proteins measured. Our strategy aims to identify lead compounds that specifically kill test cells with activated Akt signaling (yellow cells) and that spare the otherwise isogenic control cells (cyan cells).</p

The viability of BaFiso cell lines in the absence of IL-3.

<p>(A) Parental Ba/F3 derived BY and BC cells, and the BaFiso cell lines BYA and BCS were maintained in the presence or absence of IL-3 (+IL3 and −IL3). Photos were taken 24 h after transferring the cells to medium without IL-3 or in the presence of 3 ng/ml of the recombinant cytokine. (B) Measurement of cell viability using the Alamar blue assay. Alamar blue fluoresces and changes color in response to chemical reduction, and the extent of the conversion is a reflection of cell viability. Metabolic conversion of Alamar blue to its reduced, pink derivative upon cytokine-deprivation (−IL-3) or in its presence (+IL-3). (C), Bar graph showing the results of Alamar Blue cell viability assay. Maximal absorbance of the reduced and oxidized forms of AlamarBlue™, 570 and 600 nm was measured using Victor 1420 multilabel counter 24 h after IL-3 withdrawal. The percentage of cell survival was calculated compared with control cells in the presence of 3 ng/ml of IL3. The data represents three independent experiments performed in triplicate samples. (D) Time course of cell viability upon IL-3 withdrawal. Cells were washed twice in PBS and seeded in media lacking IL-3. Viability was assessed at 12 hour intervals by trypan blue exclusions followed by cell countings. Black rhombs and open squares represent percentage viability of BY and BC cells, respectively. Open triangles and black circles represent percentage viability of BYA and BCS cells, respectively. Data are presented as mean±SD from three independent experiments.</p

The analysis of Akt phosphorylation in BaFiso cell lines.

<p>Immunoblot analysis of total lysates from the Ba/F3 derived cell lines BY, BC, BYA and BCS. Cells were seeded, grown to 80% confluence and starved for 12 h in IL-3 free medium (−IL-3) or maintained in medium containing 3 ng/ml of the recombinant cytokine (+IL-3). Relevant proteins are indicated by arrows in the blot from a representative experiment.</p

Phenotypic effect of hormone treatment.

<p><b>A and B</b>) <b>Inflammation incidence in hormone- treated mice.. </b><b>A</b>) H&E staining of a prostate of a 16-week-old tgPim1/PTEN-Het mouse after one round of hormone treatment showing inflammation and micro-abscesses (black arrows). <b>B</b>) Percentage of inflammation incidence in every genotype after one or 2 rounds of hormone treatment, respectively. <b>C and D</b>) <b>Pyelonephritis incidence in hormone-treated mice.. </b><b>C</b>) c1 and c2: H&E staining (augmentation 0.5x, panoramic viewer) of a healthy kidney from a 24 week-old WT mouse vs. a kidney displaying pyelonephritis from a 24-week-old tgPim1 mouse, both after 2 rounds of hormone treatment. Note pelvic cystic dilatation (#) with narrowing of the remaining parenchyma (*). c3 and c4: H&E staining of the same kidneys (augmentation 25x). Note the well-demarcated areas of renal infarct in the animal with pyelonephritis, in which more than one-third of the parenchyma is affected. <b>D</b>) Percentage of pyelonephritis incidence in every genotype after one or 2 rounds of hormone treatment, respectively.</p

KI67 levels in mPIN lesions.

<p>Low grade: mPIN I and mPIN II. High grade: mPIN III and mPIN IV. In yellow conditions in which senescence markers were observed. NL: No lesions observed; NA: Not assessed.</p

Senescence in prostate lesions.

<p>To determine senescence in prostate lesions, immunohistochemistry for several senescence markers, such as p21, p16 and p19, was performed in the prostate tissues of mice showing high grade and low grade mPIN lesions (10-month-old untreated mice and 16-week-old hormone treated mice). <b>A</b>) Senescence markers in prostate lesions. To determine senescence in prostate lesions from 16-week-old mice before and after hormone treatment, as well in 10-month-old untreated mice, immunohistochemistry for several senescence markers, namely p21, p16 and p19, was performed on the prostate tissues of mice of each genotype using the following grading scale for senescence grade (s-grade): s-grade 1 - few cells in 1 lesion (1–5% positive cells); s-grade 2 - few cells in more than one lesion; s-grade 3 - several cells (5–20%) in more than one lesion; s-grade 4 - more than 20% positive cells in more than one lesion; only concluding true senescence if s-grades 3 or 4 were reached in the same animal for at least 2 senescence markers preferably p16 and p19. <b>B</b>) Representative picture showing that senescence markers appear only in high grade mPIN in untreated mice. <b>C</b>) Senescence markers in high grade lesions – aging vs. hormone treatment. Immunohistochemistry for p16 and p19 in high grade mPIN lesions (mPINIV) in a 10-month-old untreated PTEN-Het mouse and a 16-week-old hormone treated PTEN-het mouse.</p

characteristics of the PIM1 conditional transgenic mice.

<p><b>A</b>) Schematic representation of the transgene carrying Pim1. The arrow indicates the generation of tissue specific transgenic mice expressing PIM1 by crossing with mice expressing Cre-recombinase under a tissue specific promoter; the Lox/Stop/Lox cassette is excised allowing transcription. <b>B</b>) Expression of PIM1 in Pim1/PSA-Cre mice. RNA was extracted from different tissues of 10-week-old mice and the specific transgenic <i>Pim1</i> transcription analyzed by RT-PCR. <b>C</b>) Levels of human and mouse PIM1 mRNAs calculated by quantitative RT-PCR. Graph shows average (± SD) levels of expression in the prostate of human (black) or mouse (grey) PIM1 mRNA of at least two mice per genotype performed in triplicate. Data were normalized to 1 (log 10 = 0) using the levels of mouse PIM1 levels in wild type animals. ND: Not detected. Human PIM1 was not detected in WT nor PTEN-Het mice. <b>D</b>) Level of PIM1 protein in the mice of different genotypes measured by Western blot. The prostate of 2 animals (1 and 2) per cohort was processed to extract total proteins and run in a PAGE to allow further identification by western blot. Transgenic PIM1 was identified by myc-tag present in the transgene. Total PIM1 content was identified with a PIM1 antibody cross-reacting with human and mouse species. Activity was measured as Bad phosphorylation levels at S112. <b>E</b>) Representative pictures of high grade mPIN lesions developed after hormone treatment. To determine the development of mPIN lesions due to hormone treatment, 8-week-old untreated and hormone-treated (for 1 or 2 rounds) mice of each genotype were sacrificed and prostate tissue was obtained (see text for details). H&E staining of prostate tissue was used for mPIN grading. Pictures represent: No treatment: wt) pl-grade 0; tgPim1) pl-grade 1; PTEN-Het) pl-grade 5; tgPim1/PTEN-Het) pl-grade 8; 1 round of hormone treatment: wt) pl-grade 2; tgPim1) pl-grade 4; PTEN-Het) pl-grade 10; tgPim1/PTEN-Het) pl-grade 11; 2 rounds of hormone treatment: wt) pl-grade 6; tgPim1) p-grade 13.</p

mPIN lesions in 10-month-old untreated mice.

<p>To determine the development of mPIN lesions over time without hormone treatment, 10-month-old untreated mice of each genotype were sacrificed and prostate tissue was obtained. H&E staining of prostate tissue was used for mPIN grading, applying the Bar Harbor grading system, and subdivided into mPIN I-IV as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060277#pone.0060277.s006" target="_blank">Table S6</a>. <b>A</b>) Representative picture of the maximum grade reached: wt) grade 6; tgPim1) grade 5; PTEN-Het) grade 13; tgPim1/PTEN-Het) grade 11. <b>B</b>) Incidence (in %) of mPIN lesions per genotype in 10-month-old mice. Percentage of developed mPIN grade (mPIN I-IV and microinvasive carcinoma) was determined for each genotype using H&E staining of prostate tissue. <b>C</b>) mPIN lesions in 10-month-old untreated mice. The graphs represent the grading observed in the different prostates analyzed; statistical relevance is also shown: * = <0.05; ** = <0.01; *** = <0.001. <b>D:</b> H&E staining of a microcarcinoma. Microcarcinoma in a 10-month-old PTEN-Het mouse.</p