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

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

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 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