Journal: Cancer Cell

Article Title: Hoxa9 and Meis1 Cooperatively Induce Addiction to Syk Signaling by Suppressing miR-146a in Acute Myeloid Leukemia

doi: 10.1016/j.ccell.2017.03.001

Figure Lengend Snippet: Meis1 Increases Syk Protein Levels in Hoxa9-Driven Leukemia (A) Kaplan-Meier survival curves of mice transplanted with either H- or H/M-transformed myeloid progenitor cells (n = 11). The p value is from a Mantel-Cox test. (B) Volcano plot relating q values for differential protein expression to average normalized SILAC ratios from six biological replicates. Blue (higher expression in H cells) and orange (higher expression in H/M cells) dots indicate significantly regulated proteins (q < 0.01). (C) Heatmap of SILAC ratios for significantly differentially expressed proteins in H and H/M cells across the six biological replicates. (D) Syk protein expression in H and H/M cells by immunoblotting. Actin was used as loading control for relative protein quantification. (E) Relative Syk mRNA expression as measured by qPCR, normalized to GAPDH expression (mean ± SD, n = 3); ns, not significant (two-sided unpaired t test). (F and G) Immunohistochemical staining of HOXA9, MEIS1, and SYK in bone marrow biopsies from patients with AML. SYK expression levels were analyzed in 21 AML cases with high HOXA9 expression (F) and 28 cases with high HOXA9/MEIS1 expression (G). Proportions of SYK expression levels as determined by two independent pathologists using a three-stage staining score are shown. See also Figure S1 , , and .

Article Snippet: CD34+ BM-MNCs were isolated using the human CD34 MultiSort Kit (Miltenyi Biotec) following the manufacturer's instructions.

Techniques: Transformation Assay, Expressing, Multiplex sample analysis, Western Blot, Control, Immunohistochemical staining, Staining

Journal: Cancer Cell

Article Title: Hoxa9 and Meis1 Cooperatively Induce Addiction to Syk Signaling by Suppressing miR-146a in Acute Myeloid Leukemia

doi: 10.1016/j.ccell.2017.03.001

Figure Lengend Snippet: Meis1 Sensitizes Hoxa9-Driven Leukemia to Syk Inhibition (A) Syk protein expression in H/M cells transfected with either a control shRNA (GL2) or two shRNAs targeting Syk. Actin was used as loading control for relative protein quantification. (B) Percentage of BFP-positive shRNA-expressing cells relative to BFP-negative shRNA-negative cells at the times indicated (mean ± SD, normalized to day 0, n = 3). (C) Same as (A), before and after 5 days of doxycycline (dox) treatment in vivo. (D) Kaplan-Meier survival curves of mice transplanted with H/M cells and treated with doxycycline for 43 days to express non-specific control and Syk-specific shRNA (n = 8). The p value is from a Mantel-Cox test. (E) Percentage of YFP-positive cells from peripheral blood of mice transplanted with H (left) or H/M (right) cells after treating for 7 days with R788 or placebo. Measurements were taken at the indicated time points. The black line connects median values. (F) Kaplan-Meier survival curves of mice transplanted with either H or H/M cells and treated for 20 days with R788 or placebo (n = 11). The p value is from a Mantel-Cox test. (G) Relative HOXA9 and MEIS1 mRNA expression in MV4-11 and KG1 cell lines, and in patient-derived AML cells as measured by qPCR, normalized to GAPDH expression (mean ± SD, n = 3). (H) (p)SYK expression in the patient-derived AML cells in (G). Actin was used as loading control for relative protein quantification. avg, average. (I) Half maximal inhibitory concentration (IC 50 ) for R406 (left) and PRT062607 (right) in patient-derived AML cells as determined by an Annexin V/7-AAD apoptosis assay. Cells were treated for 24 hr and DMSO was used as a control (n = 3). Representative dose-response curves for AML no. 1 (HOXA9 high, MEIS1 low) and AML no. 5 (HOXA9 high, MEIS1 high) are shown at the top. Ticks correspond to estimated IC 50 values. (J) Relative viability of CD34 + bone marrow cells from healthy donors. Cells were treated with either R406 or PRT062607. Blue lines indicate the IC 50 for both SYK inhibitors in H cells. (K) Kaplan-Meier survival curves of NSG mice transplanted with patient-derived AML cells indicated in (G) and treated for 14 days with R788 or vehicle (n = 6 for AML no. 1 and 5; n = 5 for AML no. 2 and 6). The p values are from a Mantel-Cox test. See also Figure S6 .

Article Snippet: CD34+ BM-MNCs were isolated using the human CD34 MultiSort Kit (Miltenyi Biotec) following the manufacturer's instructions.

Techniques: Inhibition, Expressing, Transfection, Control, shRNA, In Vivo, Derivative Assay, Concentration Assay, Apoptosis Assay

Journal: Cancer Cell

Article Title: Hoxa9 and Meis1 Cooperatively Induce Addiction to Syk Signaling by Suppressing miR-146a in Acute Myeloid Leukemia

doi: 10.1016/j.ccell.2017.03.001

Figure Lengend Snippet:

Article Snippet: CD34+ BM-MNCs were isolated using the human CD34 MultiSort Kit (Miltenyi Biotec) following the manufacturer's instructions.

Techniques: Recombinant, Blocking Assay, Lysis, SYBR Green Assay, Reporter Assay, Extraction, Bicinchoninic Acid Protein Assay, cDNA Synthesis, Reverse Transcription, TaqMan microRNA Assay, Mass Spectrometry, Microarray, Gene Expression, Retroviral, Negative Control, Plasmid Preparation, Software, Multiplex sample analysis

Journal: Cell Reports

Article Title: Chromatin accessibility governs the differential response of cancer and T cells to arginine starvation

doi: 10.1016/j.celrep.2021.109101

Figure Lengend Snippet:

Article Snippet: For microarray analysis, RNA was extracted from THP1 or stimulated human CD4+ T cells using the RNeasy Mini kit (QIAGEN).

Techniques: Recombinant, Multiplex sample analysis, Cell Isolation, Activation Assay, Staining, Flow Cytometry, Expressing, Reverse Transcription, Transfection, TA Cloning, Plasmid Preparation, Methylation, Immunoprecipitation, Purification, DNA Library Preparation, Library Quantification, Control, Sequencing, Methylation Sequencing, Amplification, Software

Journal: BMC Medical Genomics

Article Title: Identification of protein-coding and non-coding RNA expression profiles in CD34 + and in stromal cells in refractory anemia with ringed sideroblasts

doi: 10.1186/1755-8794-3-30

Figure Lengend Snippet: Validation of gene expression data of CD34 + cells by real-time RT-PCR . Comparison of gene expression levels by real-time RT-PCR (black bars) and microarray experiments (gray bars) for eight select genes (names indicated at bottom) in CD34 + cells of MDS-RARS patients and healthy individuals. Positive and negative fold change values indicate the up- or down-regulation of expression in MDS-RARS patients in relation to the expression in healthy individuals, respectively.

Article Snippet: Data were normalized among the samples by quantile [ ] using Spotfire DecisionSite ® for Microarray Analysis (TIBCO Software Inc, Somerville, MA, USA).

Techniques: Biomarker Discovery, Gene Expression, Quantitative RT-PCR, Comparison, Microarray, Expressing

Journal: BMC Medical Genomics

Article Title: Identification of protein-coding and non-coding RNA expression profiles in CD34 + and in stromal cells in refractory anemia with ringed sideroblasts

doi: 10.1186/1755-8794-3-30

Figure Lengend Snippet: Validation of gene expression data of stromal cells by real-time RT-PCR . Comparison of gene expression obtained from real-time RT-PCR (black bars) and microarray experiments (gray bars) for four select genes (names indicated at bottom) in stromal cells of MDS-RARS patients and of healthy individuals. The positive and negative fold change values indicate up- or down-regulation of expression in MDS-RARS patients in relation to the expression in healthy individuals, respectively.

Article Snippet: Data were normalized among the samples by quantile [ ] using Spotfire DecisionSite ® for Microarray Analysis (TIBCO Software Inc, Somerville, MA, USA).

Techniques: Biomarker Discovery, Gene Expression, Quantitative RT-PCR, Comparison, Microarray, Expressing

Journal: British Journal of Cancer

Article Title: SILAC-based phosphoproteomics reveals an inhibitory role of KSR1 in p53 transcriptional activity via modulation of DBC1

doi: 10.1038/bjc.2013.628

Figure Lengend Snippet: Identification of KSR1-regulated phosphoproteome in breast cancer cells. ( A ) Experimental schematic outline of SILAC experiment. ( B ) Scatter plot comparison of phosphosite ratios quantified from control vs KSR1-overexpressed MCF7 cells. ( C ) Gene ontology (GO) Classification of the KSR1-regulated phosphoproteome in MCF7 cells according to molecular functions, biological processes and cellular compartmentalisation.

Article Snippet: The following antibodies were used: KSR1 rabbit polyclonal from Cell Signaling (Hitchin, UK), anti-Flag mouse monoclonal (Sigma Aldrich), p53 mouse monoclonal DO-1 from Santa Cruz (Wiltshire, UK), acetylated-p53 and phospho-p53 Ser15 rabbit polyclonal (Cell Signaling), SIRT1 rabbit polyclonal (Santa Cruz), DBC1 and phospho-DBC1 Thr454 rabbit polyclonal (Cell Signaling) and β -actin mouse monoclonal from Abcam (Cambridge, UK).

Techniques: Multiplex sample analysis, Comparison, Phospho-proteomics, Control

Journal: British Journal of Cancer

Article Title: SILAC-based phosphoproteomics reveals an inhibitory role of KSR1 in p53 transcriptional activity via modulation of DBC1

doi: 10.1038/bjc.2013.628

Figure Lengend Snippet: Effects of KSR1 on p53 transcriptional activity in the presence or absence of etoposide by luciferase assays. ( A ) MCF7 cells were transiently co-transfected with either pCMV6 (vector) or pCMV6-KSR1 plasmids in the presence of four individual p53-dependent promoter constructs expressing firefly luciferase genes (p53-R2, p53-AIP1, p53-CYCLIN G1 and p53-IGFBP3) following dimethylsulphoxide (DMSO) or etoposide (40 μ M ) treatment for 3 h. ( B ) MCF7 cells were transfected with control siRNA (siCT) or siKSR1 for 48 h, followed by transfection of three p53-dependent promoter constructs expressing firefly luciferase genes (p53-R2, p53-AIP1 and p53-CYCLIN G1) for additional 24 h. DMSO or etoposide (40 μ M ) were subsequently added as described above. Firefly luciferase activity was measured (renilla luciferase activity was used to normalise transfection efficiency). The normalised luciferase activity of empty vector is set as 1. Results shown are the average of at least three independent experiments and error bars represent s.d. Student's t -test was performed using SPSS 16.0 statistical software (SPSS Inc.). (* P <0.05, ** P <0.01).

Article Snippet: The following antibodies were used: KSR1 rabbit polyclonal from Cell Signaling (Hitchin, UK), anti-Flag mouse monoclonal (Sigma Aldrich), p53 mouse monoclonal DO-1 from Santa Cruz (Wiltshire, UK), acetylated-p53 and phospho-p53 Ser15 rabbit polyclonal (Cell Signaling), SIRT1 rabbit polyclonal (Santa Cruz), DBC1 and phospho-DBC1 Thr454 rabbit polyclonal (Cell Signaling) and β -actin mouse monoclonal from Abcam (Cambridge, UK).

Techniques: Activity Assay, Luciferase, Transfection, Plasmid Preparation, Construct, Expressing, Control, Software

Journal: British Journal of Cancer

Article Title: SILAC-based phosphoproteomics reveals an inhibitory role of KSR1 in p53 transcriptional activity via modulation of DBC1

doi: 10.1038/bjc.2013.628

Figure Lengend Snippet: Effects of KSR1 on p53 mRNA, total protein and neddylation levels and on p53 subcellular localisation. ( A ) Effects on p53 mRNA and total protein levels after KSR1 overexpression. MCF7 cells were transiently transfected with pCMV6 or pCMV6-KSR1 plasmids for 24 h. Subsequently, relative mRNA levels of TP53 and p53 total protein were measured by RT-qPCR and western blotting, respectively. Gene expression level from cells transfected with pCMV6 was set as 1. Results shown are the average of at least three independent experiments. Similarly, in MCF7 stably overexpressing KSR1 cells, p53 total protein was evaluated by western blot. Blots shown are representatives of at least three independent experiments. ( B ) Immunofluorescence staining of p53 cells after 24-h transfection with either pCMV6 or pCMV6-KSR1 plasmids in MCF7. p53 was detected with an anti-p53 antibody while the nucleus was stained with 4,6-diamidino-2-phenylindole (DAPI). Representative pictures of three independent experiments are shown. Subcellular fractionation assays were performed after 24-h transfection with either pCMV6 or pCMV6-KSR1 plasmids in MCF7. Tubulin and histone deacetylase 1 (HDAC1) expression served as positive normalising control for cytoplasmic and nuclear proteins respectively. Blots shown are representatives of at least three independent experiments. ( C ) Neddylation assay on p53 after KSR1 overexpression. MCF7 cells were co-transfected with HA-NEDD8 and pCMV6 or pCMV6-KSR1 plasmids as indicated. p53 was immunoprecipitated using a p53-specific antibody (DO-1) and the neddylated-p53 was detected by immunoblotting using anti-NEDD8 and anti-p53-specific antibodies. Blots shown are representatives of at least three independent experiments. Abbreviations: IgG= immunoglobulin G; IP= immunoprecipitation.

Article Snippet: The following antibodies were used: KSR1 rabbit polyclonal from Cell Signaling (Hitchin, UK), anti-Flag mouse monoclonal (Sigma Aldrich), p53 mouse monoclonal DO-1 from Santa Cruz (Wiltshire, UK), acetylated-p53 and phospho-p53 Ser15 rabbit polyclonal (Cell Signaling), SIRT1 rabbit polyclonal (Santa Cruz), DBC1 and phospho-DBC1 Thr454 rabbit polyclonal (Cell Signaling) and β -actin mouse monoclonal from Abcam (Cambridge, UK).

Techniques: Over Expression, Transfection, Quantitative RT-PCR, Western Blot, Gene Expression, Stable Transfection, Immunofluorescence, Staining, Fractionation, Histone Deacetylase Assay, Expressing, Control, Immunoprecipitation

Journal: British Journal of Cancer

Article Title: SILAC-based phosphoproteomics reveals an inhibitory role of KSR1 in p53 transcriptional activity via modulation of DBC1

doi: 10.1038/bjc.2013.628

Figure Lengend Snippet: Mechanisms of KSR1-regulated p53 transcriptional activity. ( A ) Effects on p53 acetylation and phosphorylation of DBC1 after KSR1 overexpression followed by etoposide treatment. MCF7 cells were transiently transfected with pCMV6 (vector) or pCMV6-KSR1 plasmids for 24 h. Subsequently, cells were treated with various concentrations of etoposide (20, 40, 80 μ M , 3 h). p53 acetylation and DBC1 phosphorylation at Thr454 were assessed by immunoblotting with specific antibodies as indicated. ( B ) Effects on p53 acetylation and phosphorylation of DBC1 after KSR1 silencing followed by a titration of etoposide treatment. MCF7 cells were transfected with control siRNA (siCT) or siKSR1 for 72 h followed by etoposide treatment (20, 40, 80 μ M , 3 h). p53 acetylation and DBC1 phosphorylation at Thr454 were assessed by immunoblotting with specific antibodies as indicated. ( C ) Effect of KSR1 on p53 acetylation is through DBC1. MCF7 cells were transfected with control siRNA (siCT) or siKSR1 in concordance with siCT or siDBC1 for 72 h followed by etoposide treatment (40 μ M , 3 h). Acetylated p53, DBC1 and KSR1 protein levels were assessed by immunoblotting with specific antibodies as indicated. ( D ) Effect of KSR1 on DBC1 phosphorylation is dependent on its intact kinase domain. MCF7 cells were transiently transfected with vector, wild-type KSR1 or mutant KSR1 (R502M) plasmids for 24 h followed by etoposide treatment (40 μ M , 3 h). DBC1 phosphorylation was measured by immunoblotting with specific antibody. ( E ) Interaction of DBC1 and SIRT1 after KSR1 overexpression with etoposide treatment by immunoprecipitation (IP). MCF7 cells were transiently transfected with pCMV6 or pCMV6-KSR1 plasmids for 24 h. Subsequently, cells were treated with etoposide (40 μ M , 3 h). The interactions between SIRT1 and DBC1 were detected by IP of SIRT1 or DBC1 followed by immunoblotting with DBC1 and SIRT1 antibodies respectively. Blots shown are representatives of at least three independent experiments. Quantification of blots was analysed by ImageJ software (NIH, Bethesda, MD, USA). ( F ) Schematic model illustrating the role of KSR1 on p53 transcriptional activity in breast cancer cells with (i) basal or (ii) up-regulated levels of KSR1. Abbreviation: IgG= immunoglobulin G.

Article Snippet: The following antibodies were used: KSR1 rabbit polyclonal from Cell Signaling (Hitchin, UK), anti-Flag mouse monoclonal (Sigma Aldrich), p53 mouse monoclonal DO-1 from Santa Cruz (Wiltshire, UK), acetylated-p53 and phospho-p53 Ser15 rabbit polyclonal (Cell Signaling), SIRT1 rabbit polyclonal (Santa Cruz), DBC1 and phospho-DBC1 Thr454 rabbit polyclonal (Cell Signaling) and β -actin mouse monoclonal from Abcam (Cambridge, UK).

Techniques: Activity Assay, Phospho-proteomics, Over Expression, Transfection, Plasmid Preparation, Western Blot, Titration, Control, Mutagenesis, Immunoprecipitation, Software

Journal: British Journal of Cancer

Article Title: SILAC-based phosphoproteomics reveals an inhibitory role of KSR1 in p53 transcriptional activity via modulation of DBC1

doi: 10.1038/bjc.2013.628

Figure Lengend Snippet: Effects of KSR1 silencing on breast cancer cell proliferation in vitro . SRB assays of MCF7, ZR75-1, SKBR3 and MDA231 cells after transfection with 20 n M of either siKSR1 or ‘non-targeting' siRNA (control siRNA) or vehicle (Hiperfect) for 6 days. Error bars represent s.d. of three experiements each in quintuplicates (* P <0.05, compared with control siRNA at day 6; Student's t- test).

Article Snippet: The following antibodies were used: KSR1 rabbit polyclonal from Cell Signaling (Hitchin, UK), anti-Flag mouse monoclonal (Sigma Aldrich), p53 mouse monoclonal DO-1 from Santa Cruz (Wiltshire, UK), acetylated-p53 and phospho-p53 Ser15 rabbit polyclonal (Cell Signaling), SIRT1 rabbit polyclonal (Santa Cruz), DBC1 and phospho-DBC1 Thr454 rabbit polyclonal (Cell Signaling) and β -actin mouse monoclonal from Abcam (Cambridge, UK).

Techniques: In Vitro, Transfection, Control

Journal: British Journal of Cancer

Article Title: SILAC-based phosphoproteomics reveals an inhibitory role of KSR1 in p53 transcriptional activity via modulation of DBC1

doi: 10.1038/bjc.2013.628

Figure Lengend Snippet: KSR1 expression is altered in breast cancer tissues. Oncomine analysis was performed to examine KSR1 expression in breast normal and cancer tissues using online TCGA microarray data ( www.oncomine.org ).

Article Snippet: The following antibodies were used: KSR1 rabbit polyclonal from Cell Signaling (Hitchin, UK), anti-Flag mouse monoclonal (Sigma Aldrich), p53 mouse monoclonal DO-1 from Santa Cruz (Wiltshire, UK), acetylated-p53 and phospho-p53 Ser15 rabbit polyclonal (Cell Signaling), SIRT1 rabbit polyclonal (Santa Cruz), DBC1 and phospho-DBC1 Thr454 rabbit polyclonal (Cell Signaling) and β -actin mouse monoclonal from Abcam (Cambridge, UK).

Techniques: Expressing, Microarray