Journal: PLoS ONE

Article Title: Runx Transcription Factors Repress Human and Murine c-Myc Expression in a DNA-Binding and C-Terminally Dependent Manner

doi: 10.1371/journal.pone.0069083

Figure Lengend Snippet: (A) Schematic of the human c-Myc locus 10 kb upstream of the transcriptional start site, with consensus Runx binding sites ( 5’-TGCGGT-3’ or 5’-ACCACA-3’ ) indicated as grey rectangles. The locations of the PCR primers used in ChIP analysis are indicated by arrowheads and i, ii or iii. A grey circle indicates that endogenous Runx1 occupies that site. (B) ChIP analysis using human Jurkat T cells. A 1% fraction was reserved as an input control (Input) and the remaining chromatin was immunoprecipitated with preimmune sera (Pre-immune) or anti-distal Runx1 (α-Runx1). PCR was carried out using primer sets amplifying Runx binding sites at -0.83 (i), -7.9 (ii) and -8.9 kb (iii) upstream of the human c-Myc transcriptional start site. N =3. (C) Sequence surrounding consensus Runx binding sites. Binding sites for transcription factors known to functionally or physically interact with Runx transcription factors (Ets-1, Hes-1, PU.1, LEF-1, Myb and CEBPα [ , , , , , ]) or implicated in transcriptional silencing (AP4/ZEB E-box and RAP1 [ , , ]) are identified .

Article Snippet: Immunoblot analysis was carried out as described below to probe for bound anti-distal Runx1 immunoglobulin on fixed protein beads (anti-rabbit IgG-HRP, Santa Cruz Biotechnology) and bound polyhistidine-tagged protein on unfixed beads (Tetra-His antibody from Qiagen, Valencia, CA).

Techniques: Binding Assay, Control, Immunoprecipitation, Sequencing

Journal: PLoS ONE

Article Title: Runx Transcription Factors Repress Human and Murine c-Myc Expression in a DNA-Binding and C-Terminally Dependent Manner

doi: 10.1371/journal.pone.0069083

Figure Lengend Snippet: (A) Schematic of the murine (A) c-Myc locus 10 kb upstream of the transcriptional start site with consensus Runx binding sites indicated as grey rectangles. The locations of the PCR primers used in ChIP analysis (shown in B) are indicated by arrowheads and i, ii, iii or iv. An X indicates a Runx binding site that is not occupied by endogenous Runx1; a grey circle indicates that endogenous Runx1 occupies that site. (B) ChIP analysis using primary murine splenocytes. A 1% fraction was reserved as an input control (Input) and the remaining chromatin was immunoprecipitated with preimmune sera (Pre-immune) or anti-distal Runx1 (α-Runx1). PCR was carried out using primer sets amplifying (B) Runx binding sites located at -0.9 (i), -4.3 (ii), -5.4 (iii) and -7.6 kb (iv) upstream of the murine c-Myc transcriptional start site. N =3. (C) Sequence surrounding consensus Runx binding sites. Binding sites for transcription factors known to collaborate with Runx transcription factors (Ets-1, Hes-1, PU.1, LEF-1, Myb and CEBPα [ , , , , , ]) or implicated in transcriptional silencing (AP4/ZEB E-box and RAP1 [ , , ]) are identified .

Article Snippet: Immunoblot analysis was carried out as described below to probe for bound anti-distal Runx1 immunoglobulin on fixed protein beads (anti-rabbit IgG-HRP, Santa Cruz Biotechnology) and bound polyhistidine-tagged protein on unfixed beads (Tetra-His antibody from Qiagen, Valencia, CA).

Techniques: Binding Assay, Control, Immunoprecipitation, Sequencing

Journal: PLoS ONE

Article Title: Runx Transcription Factors Repress Human and Murine c-Myc Expression in a DNA-Binding and C-Terminally Dependent Manner

doi: 10.1371/journal.pone.0069083

Figure Lengend Snippet: (A) Schematic of the structure of Runx1 and Runx1.d190. (B) 293T. cells were transfected with empty pEGFP-N1 vector (EGFPonly, left column) as a control for cytoplasmic staining, pEGFP-N1 vector containing full-length Runx1 fused in-frame to EGFP (Runx1FL, middle column) or Runx1.d190 fused in-frame to EGFP (Runx1.d190, right column). The nuclear DNA was visualized by staining with Hoescht 33342 (Nuclear, top row). Nuclear (top row) and EGFP (middle row) fluorescence are shown in isolation and merged (Merged, bottom row). (C) Relative differences in transcription between Jurkat T cells lentivirally transduced with control empty vector or vector encoding Runx1.d190 as determined by microarray analysis are shown. A complete listing of genes whose transcription is affected by Runx1.d190 in Jurkat T cells is located at http://www.ncbi.nlm.nih.gov/geo/ . (D) ChIP analysis. Chromatin was prepared from Jurkat T cells lentivirally transduced with Runx1.d190 and immunoprecipitated with preimmune sera (Pre-immune) or anti-distal Runx1 (α-Runx1). PCR was carried out using primer sets amplifying Runx1-binding sites at -0.83 (i), -7.9 (ii) and -8.9 kb (iii) upstream of the human c-Myc transcriptional start site. N =3.

Article Snippet: Immunoblot analysis was carried out as described below to probe for bound anti-distal Runx1 immunoglobulin on fixed protein beads (anti-rabbit IgG-HRP, Santa Cruz Biotechnology) and bound polyhistidine-tagged protein on unfixed beads (Tetra-His antibody from Qiagen, Valencia, CA).

Techniques: Transfection, Plasmid Preparation, Control, Staining, Fluorescence, Isolation, Transduction, Microarray, Immunoprecipitation, Binding Assay

Journal: PLoS ONE

Article Title: Runx Transcription Factors Repress Human and Murine c-Myc Expression in a DNA-Binding and C-Terminally Dependent Manner

doi: 10.1371/journal.pone.0069083

Figure Lengend Snippet: Relative differences in transcription for SERPINB2 (A) and MYC expression (B) between Jurkat T cells retrovirally transduced with control empty vector or vector encoding Runx1.d190 (Runx1.d190) as determined by quantitative RT-PCR are shown. Columns represent the mean of data from two independent experiments in which duplicates were analyzed. Error bars represent standard deviation from the mean.

Article Snippet: Immunoblot analysis was carried out as described below to probe for bound anti-distal Runx1 immunoglobulin on fixed protein beads (anti-rabbit IgG-HRP, Santa Cruz Biotechnology) and bound polyhistidine-tagged protein on unfixed beads (Tetra-His antibody from Qiagen, Valencia, CA).

Techniques: Expressing, Transduction, Control, Plasmid Preparation, Quantitative RT-PCR, Standard Deviation

Journal: PLoS ONE

Article Title: Runx Transcription Factors Repress Human and Murine c-Myc Expression in a DNA-Binding and C-Terminally Dependent Manner

doi: 10.1371/journal.pone.0069083

Figure Lengend Snippet: (A) Schematic of TAT-Runx1.d190 fusion protein. The Runx1.d190 protein lacks the TAT peptide, but has N-terminal and C-terminal polyhistidine tags. (B) Differential affinity of polyclonal antisera recognizing the N-terminus of distal Runx1 for Runx1.d190 and TAT-Runx1.d190 proteins. Histidine-tagged Runx1.d190 (lanes 2 and 3), or TAT-Runx1.d190 protein (lanes 4 and 6) adhered to nickel beads, or nickel beads alone (lanes 1 and 5) were fixed in 1% formaldeyde to simulate ChIP conditions and incubated with control Pre-immune sera (lanes 2 and 4) or anti-distal Runx1 (α-Runx1) antisera (lanes 1, 3, 5 and 6). The amount of pre-immune or anti-distal Runx1 immunoglobin associated with fixed Runx1.d190 or TAT-Runx1.d190 proteins is shown on a representative immunoblot probed with anti-immunoglobin (Anti-Ig). The amount of Runx1.d190 or TAT-Runx1.d190 proteins immobilized on the beads prior to fixation is shown on a representative immunoblot probed with anti-polyhistidine (Anti-histidine). N =3. (C) ChIP analysis of chromatin from murine splenocytes treated with 0.5 µM TAT peptide or TAT-Runx1.d190 protein and immunoprecipitated with preimmune sera (Pre-immune) or anti-distal Runx1 (α-Runx1). PCR was carried out with primer pairs amplifying consensus Runx-binding sites at -4.3 (ii), -5.4 (iii), and -7.6 (iv) kb upstream of the murine c-Myc transcriptional start site. A representative of three independent experiments is shown. (D) The PCR product yields from chromatin from TAT peptide- or TAT-Runx1.d190-treated murine splenocytes immunoprecipitated with anti-distal Runx1, relative to input, are graphed. The PCR primers used are indicated on the x-axis. P-values derived from a two-tailed t test indicating statistical significance are shown above the brackets. (E) ChIP analysis of chromatin prepared from murine splenocytes treated with 0.5 µM TAT peptide or TAT-Runx1.d190 protein and immunoprecipitated with control IgG (IgG) or anti-polyhistidine (α-Histidine). PCR was carried out with primer pairs amplifying consensus Runx-binding sites at -4.3 (ii), -5.4 (iii), and -7.6 (iv) kb upstream of the murine c-Myc transcriptional start site. N =3.

Article Snippet: Immunoblot analysis was carried out as described below to probe for bound anti-distal Runx1 immunoglobulin on fixed protein beads (anti-rabbit IgG-HRP, Santa Cruz Biotechnology) and bound polyhistidine-tagged protein on unfixed beads (Tetra-His antibody from Qiagen, Valencia, CA).

Techniques: Incubation, Control, Western Blot, Immunoprecipitation, Binding Assay, Derivative Assay, Two Tailed Test

Journal: PLoS ONE

Article Title: Runx Transcription Factors Repress Human and Murine c-Myc Expression in a DNA-Binding and C-Terminally Dependent Manner

doi: 10.1371/journal.pone.0069083

Figure Lengend Snippet: (A) Experimental design. Murine splenocytes were left untreated (Untr.), or treated with 0.5 µM TAT-Runx1.d190 protein every 2 hours for a total of 3 treatments (X3), and harvested at the 6 hour time point. Alternatively, splenocytes were given a single protein treatment of 0.5 µM TAT-Runx1.d190 protein and harvested at 0, 2, 4 or 6 hours post-treatment. (B) SYBR Green real time PCR was carried out using cDNA prepared from the samples treated as in (A). The results of 2 independent experiments are shown. Treatment conditions described in (A) are indicated on the x-axis. Bars represent standard deviation from the mean. (C) Real-time PCR was carried out using cDNA from murine splenocytes treated with 0.5 µM TAT peptide, TAT-Runx1.d190, or TAT-Runx1.d190-K167A for 4 hours. The results of three independent experiments are shown. Bars represent standard deviation from the mean. A p-value indicating statistical significance derived from a two-tailed t test is shown.

Article Snippet: Immunoblot analysis was carried out as described below to probe for bound anti-distal Runx1 immunoglobulin on fixed protein beads (anti-rabbit IgG-HRP, Santa Cruz Biotechnology) and bound polyhistidine-tagged protein on unfixed beads (Tetra-His antibody from Qiagen, Valencia, CA).

Techniques: SYBR Green Assay, Real-time Polymerase Chain Reaction, Standard Deviation, Derivative Assay, Two Tailed Test

Journal: PLoS ONE

Article Title: Runx Transcription Factors Repress Human and Murine c-Myc Expression in a DNA-Binding and C-Terminally Dependent Manner

doi: 10.1371/journal.pone.0069083

Figure Lengend Snippet: Murine splenocytes were treated with 0.5 µM TAT peptide, TAT-Runx1.d190-K167A or TAT-Runx1.d190 for 4 hours. (A, B) Representative immunoblots of whole cell lysates probed with c-Myc 9E10 antibody (top panel) or a goat polyclonal anti-actin antibody (bottom panel) are shown. (B, D) Quantification of c-Myc protein levels normalized to actin levels from immunoblots. Bars represent standard deviation from the mean. A p-value indicating statistical significance derived from a two-tailed t test is shown. N =4 (A), N =3 (B).

Article Snippet: Immunoblot analysis was carried out as described below to probe for bound anti-distal Runx1 immunoglobulin on fixed protein beads (anti-rabbit IgG-HRP, Santa Cruz Biotechnology) and bound polyhistidine-tagged protein on unfixed beads (Tetra-His antibody from Qiagen, Valencia, CA).

Techniques: Western Blot, Standard Deviation, Derivative Assay, Two Tailed Test

Journal: Cancers

Article Title: Harnessing Gene Expression Profiles for the Identification of Ex Vivo Drug Response Genes in Pediatric Acute Myeloid Leukemia

doi: 10.3390/cancers12051247

Figure Lengend Snippet: Summary of Gene Ontology and Pathway analyses. Top 3 Gene Ontology (GO), KEGG and REACTOME terms are displayed, based on p -value.

Article Snippet: Probes for RUNX1 (Hs00231079_m1), HIF1A (Hs00153153_m1), BRE (Hs01046283_m1), STAT3 (Hs00374280_m1), CLEC7A (Hs01902549_s1), and GAPDH (Hs02786624_g1) (Thermo Scientific, Waltham, MA, US) were used to quantify gene expression, according to the manufacturers’ instructions and as previously described [ ].

Techniques: Activity Assay, Sequencing, Binding Assay

Journal: Cancers

Article Title: Harnessing Gene Expression Profiles for the Identification of Ex Vivo Drug Response Genes in Pediatric Acute Myeloid Leukemia

doi: 10.3390/cancers12051247

Figure Lengend Snippet: Technical validation of gene expression levels of selected genes. Scatter plots of gene expression levels measured by microarray and qPCR for: ( A ) CLEC7A , ( B ) HIF1A , ( C ) STAT3 , ( D ) RUNX1 , and ( E ) BRE . Correlation coefficients and p -values are calculated using the Spearman’s rank correlation. ( F ) The Spearman’s rank correlation coefficients and FDRs between LC 50 of Ara C, DNR, 2-CdA, and VP16 and expression levels of the abovementioned genes, measured by qPCR in the discovery cohort.

Article Snippet: Probes for RUNX1 (Hs00231079_m1), HIF1A (Hs00153153_m1), BRE (Hs01046283_m1), STAT3 (Hs00374280_m1), CLEC7A (Hs01902549_s1), and GAPDH (Hs02786624_g1) (Thermo Scientific, Waltham, MA, US) were used to quantify gene expression, according to the manufacturers’ instructions and as previously described [ ].

Techniques: Biomarker Discovery, Gene Expression, Microarray, Expressing

Journal: Cancers

Article Title: Harnessing Gene Expression Profiles for the Identification of Ex Vivo Drug Response Genes in Pediatric Acute Myeloid Leukemia

doi: 10.3390/cancers12051247

Figure Lengend Snippet: Independent validation of expression levels measured by qPCR of selected genes in samples of 48 pediatric AML patients. Expression levels of BRE in sensitive (light blue), intermediate (white), and resistant (red) patient samples, based on 30th and 70th percentile towards ( A ) Ara C and ( B ) DNR. ( C ) Expression levels of HIF1A , according to response to DNR. ( D ) Expression levels of RUNX1 , according to response to 2-CdA. ( E ) Expression levels of CLEC7A and ( F ) HIF1A , according to the response to VP16. p -values were calculated using Wilcoxon Rank Sum test.

Article Snippet: Probes for RUNX1 (Hs00231079_m1), HIF1A (Hs00153153_m1), BRE (Hs01046283_m1), STAT3 (Hs00374280_m1), CLEC7A (Hs01902549_s1), and GAPDH (Hs02786624_g1) (Thermo Scientific, Waltham, MA, US) were used to quantify gene expression, according to the manufacturers’ instructions and as previously described [ ].

Techniques: Biomarker Discovery, Expressing