EMLP individual and population information

Population and individual plant data based on the passport information sourced from the German genebank (IPK-Gatersleben).</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

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

LOM612 compromises the viability of human cancer cell lines.

<p>(A) Breast cancer cell line MCF7, melanoma cell line A2058 and the neuroblastoma cell line SH-SY5 were seeded at a concentration of 1× 10⁴ cells/well in 200 μl and treated with compounds LOM612 and LOM621 for 72 hours with eight 2-fold serial dilutions of each compound spanning concentrations from 50μM to 0.39μM. Data is shown as mean ± SEM of three independent experiments. ****<i>P</i><0.0001 by two-way ANOVA (ns, not significant). (B) Human liver cancer cell lines HepG2 and THLE-2 (cell line derived from primary normal liver epithelial cells) were seeded at a concentration of 1× 10⁴ cells/well in 200 μl and treated with 20 different concentrations of LOM612 from 50μM to 95pM. Data is shown as mean ± SEM of three independent experiments. ****<i>P</i><0.0001 by two-way ANOVA.</p

Primary screening identifies compounds capable of inducing FOXO translocation.

<p>(A) U2fox RELOC cells were treated either with DMSO, 4nM LMB, 10μM of compound LOM 612 or compound LOM 621 for 30 min. representative images are shown. (B) Dose-response relationship of the nuclear–cytoplasmic shuttling of FOXO following LOM 612 treatment. LOM612 induces nuclear translocation in a dose dependent manner. Represented is the percentage of cells with more GFP fluorescence accumulation in nucleus than in cytoplasm. Results represent the mean of three independent experiments.</p

Synthesis of compounds 1a-c (LOM612/621/604).

<p>(a) chlorocarbonylsulphenyl chloride, <i>N</i>, <i>N</i>-dimethylurea or <i>N</i>,<i>N</i>-dibenzylurea, acetonitrile, 2h RT for <b>3a</b>: 84%, <b>3b</b>: 62%; chlorocarbonylsulphenyl chloride, benzamide, toluene, 3h, reflux <b>3c</b>: 70%; (b) 1,4-naftochinone, <b>3a</b>, xilene, 80°C, 3h, 23%; 1,4-naftochinone, <b>3b</b>, xylene, 80°C, 3h, 63%; (C) 1,4-naftochinone, <b>3c</b>, xilene, 8h, reflux, 3h, 38%; d) CAN, CH₃CN: H₂O 9:1, 1h, RT, 89%.</p

LOM612 specifically induces the nuclear translocation of endogenous FOXO proteins.

<p>(A) Compound LOM612 induces the nuclear translocation of endogenous FOXO3a and FOXO1 protein detected by using a specific antibodies after 30 min of drug exposure. (B) LOM612 does not inhibit the nuclear export. U2OS cells stably expressing nuclear export signal (NES) (Rev-NES-EGFP) reporter were treated with DMSO, LMB, LOM612 and LOM621 for 30 min. (C) LOM612 does not induce nuclear translocation of endogenous nuclear factor (NF)–κB2 protein. Representative images of the compound-treated cells using a Leica SPE confocal imaging system. Cells were seeded automatically at appropriate density in 96-well black-wall clear-bottom tissue culture plates and allowed to attach overnight. Cells were then treated with compounds for 30 min before paraformaldehyde (Rev-NES-EGFP) or methanol (FOXO3a, NFKB2) fixation and DAPI staining.</p