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sc-12-0925_sm_SupplFigure1.pdf444KFigure S1. Immunophenotype of A9M-L2 leukemic cells. Representative scattergrams (dot plots) demonstrating comparative cell surface immunophenotype of A9M-L2 leukemic and normal bone marrow cells (Control). Bone marrow cells were stained with antibodies that recognize: Sca1, Kit, Lin, Mac1, GR1, IgM, B220, CD43, CD4 and CD8. The target cells were gated based on FSC/SSC and Myeloid, T-Cell, SKL and B-cell populations are marked.
sc-12-0925_sm_SupplFigure2.pdf160KFigure S2. Altered Cellular Dynamics and Differential Gene Expression in Cre-Treated A9M-L2 cells Bone marrow cells were obtained from terminal A9M-L2 leukemic mice and cocultured with naive GP+E cells (Control Non-Infected) or those previously infected with MSCV-Cre-GFP or control vector (MSCV-GFP) virus. (a) Cells were cultured for 48 hours prior to sorting based on eGFP positivity. Transduced cells were capable of forming colonies in methylcellulose within 10 days of culture. (b) A representative bar graph demonstrating reduced colony formation following sorting of Cre-GFP expressing cells compared to control. Mean values ± S.E.M are plotted. Significant differences are denoted p ≤ 0.01 **. (c) Total RNA isolated from Cre- or GFP-Treated A9M-L2 primary cells was converted to cDNA and examined by qRT-PCR. Differential expression of candidate genes, determined by Delta CT values equivalent to ≥4-fold changes, were identified and compared to values obtained from the array analysis of A9M-L2 and normal bone marrow samples. Fourteen genes, previously identified as demonstrating reduced expression in the A9M-L2 model compared to normal bone marrow, showed a measurable increase in expression (dark grey bars) upon deletion of the HOXA9-ires-MEIS1 element. Jarid1b and Irf4 were marked as genes validated by individual assays. (d) Transduced cells were also maintained in liquid culture for up to 14 days in defined media. Images were captured at 20X using an inverted microscope (CKX41) with attached digital camera (E620) both Olympus, Essex, UK. Microscopic analysis showed increased differentiation and adhesion, indicative of a macrophage phenotype in the Cre-Treated A9M-L2 cells compared to GFP-Treated controls.
sc-12-0925_sm_SupplFigure3.pdf26KFigure S3. Comparable levels of HOXA9 and MEIS1 expression in clinical samples and murine model. Comparative bar graph obtained from quantitative RT-PCR analysis of A9M-L2 bone marrow samples, normal mouse bone marrow control (n=5 per group) and a cohort of AML patients classified based on standard prognosis adverse (n=3), favorable (n=6) or intermediate (n=15). Relative expression is displayed as log2 estimated copy number values for HOXA9 and MEIS1 (where Ct 35 = 5 copies). Mean values ± S.E.M are plotted. Comparable gene expression was observed for the Intermediate risk patient group and the A9M-L2 model as determined by the student t-test (ns for non-significant).
sc-12-0925_sm_SupplFigure4.pdf23KFigure S4 Validation of array expression changes by SYBR QPCR. Bar graph of gene expression values denoted by Delta Ct values corrected to 18SrRNA, obtained from both TaqManTM Low Density Array (Array) and individual SYBR Green-based assays (SYBR). Comparable values or similar trends for Array and SYBR assays were demonstrated in 17/21 genes examined for A9M-L2 cells relative to normal bone marrow cells (n=5 per group). Mean values ± S.E.M are plotted.
sc-12-0925_sm_SupplFigure5.pdf55KFigure S5 Functional validation of neutral/weak sscMap connections. (Left panel) a bar chart demonstrating no significant alteration in the expression of a ten gene signature (Figure 3b) in A9M-L2 cells following treatment with three small molecule inhibitors identified as having neutral/weak sscMap connectivity. The combined expression of the gene signature (estimated copy number) was obtained for vehicle control (PBS) or Tamoxifen, Thiamine and Ascorbic acid treated A9M-L2 cells and compared to Entinostat treatment. Mean values ± S.E.M of relative expression (fold of control) was plotted. Significant differences as determined by the student t-test are denoted p ≤ 0.02**, ns= non-significance. (Right Panel) Representative colony images from A9M-L2 cells treated with vehicle (PBS), Tamoxifen (1 μM), Thiamine (100 μM) or Ascorbic acid (100 μM). Leukemic colonies were stained with 1 mg/ml p-iodonitrotetrazolium violet (INT) for 16 hours and images captured by an Olympus CKX41 microscope and camera magnification 40× using an inverted microscope (CKX41) with attached digital camera (E620) both Olympus, Essex, UK.
sc-12-0925_sm_SupplFigure6.pdf117KFigure S6 Comparable effects of low dose Entinostat and Panobinostat Western blot and densitometry analysis of primary A9M-L2 cells treated with vehicle control (DMSO), Entinostat (Ent; 300 nM) or Panobinostat (Pan-7; 7 nM and Pan-25; 25 nM). Proteins were isolated 24 hours following treatment and probed with specific antibodies for Acetylated Histone-H3/H4. Representative data from three independent experiments demonstrating comparable acetylation levels are shown normalized to β-actin.
sc-12-0925_sm_SupplFigure7.pdf33KFigure S7 Short-term repopulation of NBM cells treated with Entinostat Normal CD45.1+ donor bone marrow cells were obtained by flushing femurs and tibias of C57Bl/6-Ly5.1 mice and treated with vehicle control (DMSO) or Entinostat (300 nM) for 24 hours in maintenance media containing interleukin-6, granulocyte/macrophage colony-stimulating factor, 10 ng/mL; stem cell factor, 100 ng/mL. Recipient CD45.2+ (C57Bl/6J) mice were sublethally irradiated (650 cGy) prior to transplantation with 5 × 105 treated CD45.1+ donor cells (n=4 per group). Recipient mice were housed within a specific pathogen free animal unit (QUB-BRU) for eight weeks after which they were humanely killed by CO2 asphyxiation. Bone marrow and spleen cells were obtained from the recipient mice and treated with APC conjugated anti-mouse CD45.1 and PE conjugated anti-mouse CD45.2 (both eBioscience, Hatfield, UK). Data were acquired and analyzed using an LSRII cytometer and FACSDiva Software 6.1.2 (BD Pharmingen). (a) Representative dot plots obtained from recipient mouse bone marrow or spleen samples demonstrating the percentage retention/repopulation of donor (CD45.1+) cells in the recipient (CD45.2+) background. (b) a bar graph depicting comparable low level of repopulation (∼10%) is observed for the Entinostat or DMSO treated donor cells in the bone marrow of recipient mice compared to unstained control. A higher level of repopulation (∼20%) is observed for repopulation of the spleen for both treatments. No significant difference in repopulation ability was demonstrated in the DMSO or Entinostat treated donor cells. Mean values ± S.E.M are plotted (n=4 per group).
sc-12-0925_sm_SupplFigure8.pdf41KFigure S8 Homing of NBM or A9M-L2 cells following Entinostat treatment Normal and A9M-L2 leukemic CD45.1+ bone marrow cells were obtained by flushing femurs and tibias of donor mice and treated with vehicle control (DMSO) or Entinostat (300 nM) for 24 hours in maintenance media as above. Recipient CD45.2+ (C57Bl/6J) mice were lethally irradiated (1300 cGy) prior to transplantation with 5 × 106 treated CD45.1+ donor cells (n=5 per group). Recipient mice were housed within a specific pathogen free animal unit (QUBBRU) for 24 hours after which time they were humanely killed by CO2 asphyxiation. Bone marrow and spleen cells were obtained from the recipient mice and stained with PE conjugated anti-mouse CD45.1 (Southern Biotech, Alabama, USA). Data were acquired and analyzed using an LSRII cytometer and FACSDiva Software 6.1.2 (BD Pharmingen) or FlowJo software 7.6.5 (TreeStar Inc, Ashland, OR). (a) Bar graph showing reduced bone marrow homing of NBM or A9M-L2 cells following Entinostat treatment compared to DMSO controls. The analysis shows that homing of the donor cells to the spleen is not impaired following Entinostat treatment and that a higher percentage of A9M-L2 cells home to the spleen regardless of treatment. Mean values ± S.E.M are plotted (n=5 per group). Significant differences are denoted p ≤ 0.05 *, p ≤ 0.01 ** as determined by the student t-test. (b) Overlay of representative histogram plots obtained for CD45.1 expression in recipient mouse bone marrow and spleen cells obtained from transplanted DMSO or Entinostat-treated NBM or A9M-L2 donor cells. The treated cells are compared to unstained control.
sc-12-0925_sm_SupplFigure9.pdf45KFigure S9 Induction of apoptosis following HDACi treatment To examine the effect of HDACi treatment on apoptosis, A9M-L2 cells were cultured for up to 48 hours with Entinostat (300 nM or 1 μM) or Panobinostat (7 nM or 25 nM) and apoptosis measured using the FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, San Diego, CA) as per the manufacturer's instructions. Data were acquired and analyzed using an LSRII cytometer and FACSDiva Software 6.1.2 (BD Pharmingen). (a) Bar graph demonstrating lack of Annexin V staining over DMSO controls (∼5%) in the low dose 24 hour HDACi treatments (300 nM Entinostat, 7 nM Panobinostat) but an increase in apoptosis that is both dose and time dependent peaking (>35%) with the high dose Panobinostat (25 nM) following 48 hours of treatment in liquid culture. Mean values ± S.E.M are plotted (n=4 per group). Significant differences as determined by the Student's t-test are denoted p ≤ 0.01**. (b) Representative dot plots of Annexin V and Propidium Iodide (PI) staining from the A9M cell line or primary A9M-L2 cells for the lower dose HDACi treatments (300 nM Entinostat, 7 nM Panobinostat) at 24- and 48- hour time points.
sc-12-0925_sm_SupplTable1.pdf37KTable S1. Gene expression array profiling in A9M-L2 model. Summary of the quantitative RT-PCR array profiling results of A9M-L2 model compared to normal mouse bone marrow (NBM). Immune, stem cell pluripotent and transcription factor (Biotrove) array profiles are detailed with average Ct and standard deviation values shown. Tabulated values are from duplicate experiments of five biological replicates.
sc-12-0925_sm_SupplTable2.pdf15KTable S2. Gene ontology and pathway analysis of A9M-L2 leukemia. (a) A list of genes differentially expressed significantly in the A9M-L2 leukaemias compared to normal bone marrow controls (n=5 per group) was submitted for Bioinformatics analysis using the Database for Annotation, Visualization and Integrated Discovery (DAVID) Bioinformatics Resources 6.7 and GO Enrichment Analysis Software Toolkit (GOEAST) platforms. The percentages of genes associated with a particular process or pathway are ranked and tabulated and total numbers of genes defined within a process or pathway are tabulated in parenthesis. The upper panel (light grey) indicates statistically significant association with biological processes (DAVID) the lower panel (dark grey) with pathway associations (Kyoto Encyclopedia of Genes and Genomes, KEGG). (b) A table of process-associated genes identified in both the GOEAST and DAVID platforms. The subset of processes examined includes cell proliferation, cell activation, apoptosis, and transcription. Genes in bold font are common to both platforms demonstrating a level of robustness in the analysis.
sc-12-0925_sm_SupplTable3.pdf14KTable S3. Gene ontology of differentially expressed genes in A9M-L2 cells. Gene Ontology Enrichment Analysis Software Toolkit (GOEAST) was used to cluster molecular function (white), biological processes (light grey) and chemical component characteristics (dark grey) from the A9M-L2 gene lists obtained from array profiling experiments. The number of genes associated with a particular process and statistical measures of the possibility that the associations were made by chance (log of odds ratio of enrichment and p values) are tabulated. High log values (≥ 1.5) and low p values ≤ 0.05) indicate the GOterm associations are unlikely to be due to chance.
sc-12-0925_sm_SupplTable4.pdf13KTable S4. Genes and Probeset IDs used for sscMap Analysis Differentially expressed genes were identified from the comparative analysis of the mouse model (A9M-L2 v NBM) and patient cohort MILE data (MILE 13 v MILE 18) using two-sample t test analysis. A gene-signature with 47 Affymetrix probeset IDs, representing 30 individual genes, provided the minimum set of genes which returned significant connections to drugs at 1% false discovery rate (FDR=0.01). For the MILE study, the Affymetrix MAS5 value on the log scale was used as a measure of gene expression level. A gene signature with 24 Affymetrix probeset IDs, representing 21 individual genes, was constructed which returned significant connections to drugs at 1% false discovery rate (FDR=0.01).

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