Journal: Oncogene

Article Title: Functional characterization of the promoter region of the human EVI1 gene in acute myeloid leukemia: RUNX1 and ELK1 directly regulate its transcription.

doi: 10.1038/onc.2012.222

Figure Lengend Snippet: Figure 4. Knockdown experiments of RUNX1 and ELK1 in the HEL and F36-P cell lines. (a) Western blot analysis of total proteins with anti-RUNX1 and anti-EVI1, and quantification of the protein changes at 36 h in the HEL cell line. (b) Detection of the EVI1-1A, EVI1-1B and EVI1-1D mRNA transcripts, normalized to GAPDH expression at 24 h (calculated according to the 2-DDCt method) in the HEL cell line. (c) Western blot analysis of nuclear proteins with anti-ELK1 and anti- EVI1, and quantification of the protein changes at 36 h in HEL. (d) Detection of the EVI1-1A, EVI1-1B and EVI1-1D mRNA transcripts, normalized to GAPDH expression at 24 h (calculated according to the 2-DDCt method) in HEL. (e) Western blot analysis of total proteins with anti-RUNX1 and anti-EVI1, and quantification of the protein changes at 36 h in the F36-P cell line. (f) Detection of the EVI1-1A, EVI1-1B and EVI1-1D mRNA transcripts, normalized to GAPDH expression at 24 h (calculated according to the 2-DDCt method) in F36-P. (g) Western blot analysis of nuclear proteins with anti-ELK1 and anti-EVI1, and quantification of the protein changes at 36 h in F36-P. (h) Detection of the EVI1-1A, EVI1-1B and EVI1-1D mRNA transcripts, normalized to GAPDH expression at 24 h (calculated according to the 2-DDCt method) in F36-P (*Po0.05; **Po0.01).

Article Snippet: To study the effect of overexpression of RUNX1 or ELK1 on the EVI1 promoter, 2 106 HEL cells were transfected with 5 mg of WT or mutant 318 bp construct, with 500 ng RUNX1 expression plasmid (pFlagCMV2– AML1B (plasmid 12 504, Addgene, Cambridge, MA, USA)35) or 2.5mg ELK1 expression plasmid (pCDNA3-ELK1, kindly provided by Dr Hipskind) or the empty vectors in addition to 100 ng of pRL-SV40.

Techniques: Knockdown, Western Blot, Expressing

Journal: Oncogene

Article Title: Functional characterization of the promoter region of the human EVI1 gene in acute myeloid leukemia: RUNX1 and ELK1 directly regulate its transcription.

doi: 10.1038/onc.2012.222

Figure Lengend Snippet: Figure 5. Overexpression experiments of RUNX1 in the HEL and HL-60 cell lines. (a) Western blot analysis of total proteins with anti-RUNX1 and anti-EVI1, and quantification of the protein changes at 24 h in the HEL cell line. (b) Detection of the EVI1-1A, EVI1-1B and EVI1-1D mRNA transcripts normalized to GAPDH expression at 24 h (calculated according to the 2-DDCt method) in the HEL cell line. (c) Detection of EVI1 (EVI1 11–12 assay) normalized to HPRT expression 24 h after the transfection of RUNX1 in the HL-60 cell line (calculated according to the 2-DDCt method). (d) Luciferase assays in the HEL cell line, cotransfecting the RUNX1 expression vector (or empty vector) with WT or with the RUNX1- binding site mutated. Results represent relative Firefly/Renilla luciferase activities considering the empty vector as 1. (e) qRT–PCR showing the enrichment of acetylated histones H3/H4 on the EVI1 promoter 12 h after overexpressing RUNX1. Results were calculated using the 2-DDCt method and are presented as the fold enrichment of chromatin DNA precipitated by the specific antibody versus chromatin DNA precipitated by no antibody, as control. Values are mean þ s.e.m. from three independent experiments (*Po0.05).

Article Snippet: To study the effect of overexpression of RUNX1 or ELK1 on the EVI1 promoter, 2 106 HEL cells were transfected with 5 mg of WT or mutant 318 bp construct, with 500 ng RUNX1 expression plasmid (pFlagCMV2– AML1B (plasmid 12 504, Addgene, Cambridge, MA, USA)35) or 2.5mg ELK1 expression plasmid (pCDNA3-ELK1, kindly provided by Dr Hipskind) or the empty vectors in addition to 100 ng of pRL-SV40.

Techniques: Over Expression, Western Blot, Expressing, Transfection, Luciferase, Plasmid Preparation, Binding Assay, Quantitative RT-PCR, Control

Journal: Oncogene

Article Title: Functional characterization of the promoter region of the human EVI1 gene in acute myeloid leukemia: RUNX1 and ELK1 directly regulate its transcription.

doi: 10.1038/onc.2012.222

Figure Lengend Snippet: Figure 6. RUNX1, ELK1 and GATA1 associate with each other in the promoter region of EVI1. (a) ChIP assay performed in the HEL and HEK293T cell lines. qRT–PCR was performed on fragmented chromatin precipitated by Normal rabbit IgG (ELK1) or Normal goat IgG (RUNX1, GATA1), or the specific anti-RUNX1, anti-ELK1 or anti- GATA1 antibodies. The promoter region of GAPDH was used as a negative region, and cFOS or GATA2 promoter regions as positive controls for ELK1 or GATA1 binding, respectively. qRT–PCR results were calculated using the 2-DDCt method, and they are presented as the fold enrichment of chromatin DNA precipitated by the specific antibody versus chromatin DNA precipitated by normal IgG, as control. (b) Protein immunoprecipitation assay using anti-RUNX1, anti-ELK1 and anti-GATA1 or non-specific IgGs used as a control. Then, the immunoprecipitates were analyzed for the presence of RUNX1, ELK1 or GATA1.

Article Snippet: To study the effect of overexpression of RUNX1 or ELK1 on the EVI1 promoter, 2 106 HEL cells were transfected with 5 mg of WT or mutant 318 bp construct, with 500 ng RUNX1 expression plasmid (pFlagCMV2– AML1B (plasmid 12 504, Addgene, Cambridge, MA, USA)35) or 2.5mg ELK1 expression plasmid (pCDNA3-ELK1, kindly provided by Dr Hipskind) or the empty vectors in addition to 100 ng of pRL-SV40.

Techniques: Quantitative RT-PCR, Binding Assay, Control, Immunoprecipitation

Journal: Oncogene

Article Title: Functional characterization of the promoter region of the human EVI1 gene in acute myeloid leukemia: RUNX1 and ELK1 directly regulate its transcription.

doi: 10.1038/onc.2012.222

Figure Lengend Snippet: Figure 7. Luciferase assays of the 318-bp region in the HEL and Kasumi-1 cell lines with the WT construct, or with the RUNX1 and/or ELK1- binding sites mutated. Results represent relative Firefly/Renilla luciferase activities considering the empty pGL3basic as 1. Values are the mean þ s.e.m. from at least three independent experiments (*Po0.05; **Po0.01).

Article Snippet: To study the effect of overexpression of RUNX1 or ELK1 on the EVI1 promoter, 2 106 HEL cells were transfected with 5 mg of WT or mutant 318 bp construct, with 500 ng RUNX1 expression plasmid (pFlagCMV2– AML1B (plasmid 12 504, Addgene, Cambridge, MA, USA)35) or 2.5mg ELK1 expression plasmid (pCDNA3-ELK1, kindly provided by Dr Hipskind) or the empty vectors in addition to 100 ng of pRL-SV40.

Techniques: Luciferase, Construct, Binding Assay

Journal: Oncogene

Article Title: Functional characterization of the promoter region of the human EVI1 gene in acute myeloid leukemia: RUNX1 and ELK1 directly regulate its transcription.

doi: 10.1038/onc.2012.222

Figure Lengend Snippet: Figure 8. Western blot analysis of EVI1 and RUNX1 in 16 AML patient samples (eight with EVI1 overexpression). Quantification of the relative protein level of EVI1 or RUNX1 and ACTIN is shown.

Article Snippet: To study the effect of overexpression of RUNX1 or ELK1 on the EVI1 promoter, 2 106 HEL cells were transfected with 5 mg of WT or mutant 318 bp construct, with 500 ng RUNX1 expression plasmid (pFlagCMV2– AML1B (plasmid 12 504, Addgene, Cambridge, MA, USA)35) or 2.5mg ELK1 expression plasmid (pCDNA3-ELK1, kindly provided by Dr Hipskind) or the empty vectors in addition to 100 ng of pRL-SV40.

Techniques: Western Blot, Over Expression

Journal: Oncogene

Article Title: Functional characterization of the promoter region of the human EVI1 gene in acute myeloid leukemia: RUNX1 and ELK1 directly regulate its transcription.

doi: 10.1038/onc.2012.222

Figure Lengend Snippet: Figure 9. Role of EVI1 and RUNX1 in megakaryocytic differentiation. (a) Detection of EVI1, RUNX1, ELK1 and the megakaryocytic markers, ITGA2B and ITGB3 expression at 48 h in response to TPA, normalized to HPRT expression (calculated according to the 2-DDCt method). (b) Western blot analysis of total proteins with anti-EVI1, anti-ELK1 and anti-RUNX1 after 48 h of the respective knock down and TPA treatment. (c) Detection of EVI1 11–12, ELK1, RUNX1 and the megakaryocytic markers, ITGA2B and ITGB3 expression after the indicated knock down and 48 h after the TPA treatment, normalized to HPRT expression (calculated according to the 2-DDCt method). (d) ChIP assay in K562 cell line before and after TPA treatment. qRT–PCR was performed on fragmented chromatin precipitated by Normal rabbit IgG (ELK1) or Normal goat IgG (RUNX1, GATA1), or the specific anti-RUNX1, anti-ELK1 or anti-GATA1 antibodies. The promoter region of GAPDH was used as a negative region, and LMO2 or cFOS promoter regions as positive controls for RUNX1 and GATA1 or ELK1 binding, respectively. PCR results were calculated using the 2-DDCt method, and they are presented as the fold enrichment of chromatin DNA precipitated by the specific antibody versus chromatin DNA precipitated by normal IgG, as control (*Po0.05; **Po0.01; ***Po0.001).

Article Snippet: To study the effect of overexpression of RUNX1 or ELK1 on the EVI1 promoter, 2 106 HEL cells were transfected with 5 mg of WT or mutant 318 bp construct, with 500 ng RUNX1 expression plasmid (pFlagCMV2– AML1B (plasmid 12 504, Addgene, Cambridge, MA, USA)35) or 2.5mg ELK1 expression plasmid (pCDNA3-ELK1, kindly provided by Dr Hipskind) or the empty vectors in addition to 100 ng of pRL-SV40.

Techniques: Expressing, Western Blot, Knockdown, Quantitative RT-PCR, Binding Assay, Control