Journal: PLoS ONE

Article Title: Transcriptional Repression of Cdc25B by IER5 Inhibits the Proliferation of Leukemic Progenitor Cells through NF-YB and p300 in Acute Myeloid Leukemia

doi: 10.1371/journal.pone.0028011

Figure Lengend Snippet: A. RT-PCR was performed to analyze IER5 mRNA expression in AML cell lines (KG-1, Kasumi-1, U937, and YRK2) treated with or without TMPP (5 and 10 µM) or Ara-C (1 µM) for 24 h. GAPDH mRNA expression is shown as an internal control. RT-PCR results representative of three independent experiments are shown. Relative levels of IER5 mRNA expression in AMLs were measured using QRT-PCR. B. IER5 mRNA and protein expression in U937 cells that were transfected with IER5 cDNA, shRNA-#1, or -#2, or were treated with TMPP (5 µM), were assessed using RT-PCR and QRT-PCR, and Western blotting, respectively. Actin was immunoblotted as a loading control. The expression levels of the target mRNAs were normalized to the expression of GAPDH mRNA. The results are expressed relative to the untreated control which was set at 1. Each RT-PCR assay was performed at least three times, and the results are expressed as means ± SD. * P <0.01. Western blotting results representative of three independent experiments are shown.

Article Snippet: Full-length cDNAs encoding human IER5 and Cdc25B were obtained by RT-PCR using human bone marrow cDNA (BD Biosciences Clontech, Palo Alto, CA) as a template and were cloned into the eukaryotic expression vector pcDNA3.1/V5-His (Invitrogen, Carlsbad, CA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Quantitative RT-PCR, Transfection, shRNA, Western Blot

Journal: PLoS ONE

Article Title: Transcriptional Repression of Cdc25B by IER5 Inhibits the Proliferation of Leukemic Progenitor Cells through NF-YB and p300 in Acute Myeloid Leukemia

doi: 10.1371/journal.pone.0028011

Figure Lengend Snippet: A. The cell proliferation of U937 cells, transfected with IER5 cDNA or treated with TMPP, was measured by counting cells using a hemocytometer (upper panel). Cell counting was started 3 days after transfection, and was performed every 24 h for 3 days. Data are shown as means ± S.D. of triplicate cultures and are representative of three independent experiments. * P <0.01 compared with untransfected control cells. Cell viability of the IER5 over-expressing U937 cells was assessed by counting of viable cells using trypan blue staining at 72 h, starting 3 days after DNA transfection (bottom panel). B. QRT-PCR analysis of IER5 mRNA expression in untreated cells, IER5 cDNA-transfected cells, and TMPP-treated U937 cells. QRT-PCR was started 3 days after transfection, and was performed every 24 h for 3 days. Data are shown as means ± S.D. of triplicate cultures and are representative of three independent experiments. The levels of the quantified RT-PCR products were normalized t o GAPDH expression in the same sample and were then expressed relative to the mRNA level of a normal control, which was assigned a value of 1. * P <0.01 compared with untransfected control cells. The protein expression of IER5 in cells was analyzed after 3 days of culture (bottom panels). Blotting of Actin was used as a loading control. C. The cell cycle distribution of U937 cells that were transfected with control DNA, IER5 cDNA, scrambled shRNA, IER5 shRNA #1, IER5 shRNA #1, or were treated with TMPP, was analyzed using flow cytometric analysis. The transfected or TMPP-treated U937 cells were harvested after 3 days. The fraction of cells in the G1, S and G2/M stage of the cell cycle was determined. Data are shown as means ± S.D. of triplicate cultures. The IER5 mRNA expression of the cells is shown at bottom and was assessed using RT-PCR. The RT-PCR results are representative of three independent experiments. Data are shown as means ± S.D. of triplicate cultures. * P <0.01 compared with control cells. D and E. Cell cycle analysis (D) and changes of mitochondrial membrane potential (ΔΨm) (E) in IER5 overexpressed or TMPP treated AML cells. U937 cells were transfected with IER5 cDNA or treated with TMPP (5 µM). Mitochondrial membrane potential was determined 3 days after transfection or TMPP treatment by staining of the cells with DiOC6 followed by flow cytometric analysis. The FACS results are representative of three independent experiments. NC; Negative control.

Article Snippet: Full-length cDNAs encoding human IER5 and Cdc25B were obtained by RT-PCR using human bone marrow cDNA (BD Biosciences Clontech, Palo Alto, CA) as a template and were cloned into the eukaryotic expression vector pcDNA3.1/V5-His (Invitrogen, Carlsbad, CA).

Techniques: Transfection, Cell Counting, Expressing, Staining, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, shRNA, Cell Cycle Assay, Negative Control

Journal: PLoS ONE

Article Title: Transcriptional Repression of Cdc25B by IER5 Inhibits the Proliferation of Leukemic Progenitor Cells through NF-YB and p300 in Acute Myeloid Leukemia

doi: 10.1371/journal.pone.0028011

Figure Lengend Snippet: A. U937 cells were transfected with control DNA, IER5 cDNA, or were treated with TMPP (5 µM or 10 µM). The cells were harvested after 3 days, and the expression of the indicated cell cycle regulating proteins was analyzed by SDS-PAGE followed by Western blotting using the indicated specific antibodies. Blotting of Actin was used as a loading control. B. Schematic representation of the human Cdc25B promoter regions, indicating sites for Sp1, Sp3, and NF-YB motifs, amplified from the precipitated DNA by specific primer sets 1 and 2. C. ChIP analysis for IER5 in the Cdc25B promoter. D & E. IER5-dependent changes in Sp1, Sp3 and NF-YB binding (E), and the bindings of coactivators, DNMT1 and p300, to interact with Sp1 and NF-YB, respectively (F) in Cdc25B promoter. U937 cells were either untransfected or were transfected with the control DNA or IER5 DNA. After 3 days culture, cells were cross-linked with 1% formaldehyde and ChIP was performed with either control antibody (IgG) or the anti-IER5 antibody. The precipitated DNA was then assayed by PCR using pairs (primer set 1 and 2) of oligonucleotides encompassing specific regions of the Cdc25B promoter. The values of ChIP efficiencies are given as % of input.

Article Snippet: Full-length cDNAs encoding human IER5 and Cdc25B were obtained by RT-PCR using human bone marrow cDNA (BD Biosciences Clontech, Palo Alto, CA) as a template and were cloned into the eukaryotic expression vector pcDNA3.1/V5-His (Invitrogen, Carlsbad, CA).

Techniques: Transfection, Expressing, SDS Page, Western Blot, Amplification, Binding Assay

Journal: PLoS ONE

Article Title: Transcriptional Repression of Cdc25B by IER5 Inhibits the Proliferation of Leukemic Progenitor Cells through NF-YB and p300 in Acute Myeloid Leukemia

doi: 10.1371/journal.pone.0028011

Figure Lengend Snippet: A. Selection of ALDH hi /CD34 + hematopoietic progenitor cells from the bone marrow of two healthy volunteers (#1 and #2) by FACS sorting. Region P denotes populations of ALDH hi cells. Region R and S denote populations of CD34 + and CD34 - cells in the ALDH hi population (Region P), respectively. Negative control, light grey region; CD34-PE staining, dark grey region. B. Selection of ALDH hi /CD34 + hematopoietic progenitor cells from the bone marrow of two AML patients (M1 and M2) by FACS sorting. Region P denotes populations of ALDH hi cells. Region R and S denote populations CD34 + and CD34 - cells in the ALDH hi population (Region P), respectively. Negative control, light grey region; CD34-PE staining, dark grey region. C. ALDH hi /CD34 + cells were purified from a healthy volunteer (#1) and an AML patient (M1), and were cultured in semisolid methylcellulose media. The ALDH hi /CD34 + cells from each source were left untransfected or were transfected with IER5 cDNA, or treated with TMPP (5 µM). After 14 days culture, the colony forming ability of the cells was analyzed (left upper panel) and the cells were viewed using phase-contrast microscopy. Original magnification ×4 (left bottom panels). Their mRNA expression of IER5 and Cdc25B was assessed using RT-PCR and quantitative RT-PCR (right panels). Colonies formed by these ALDH hi /CD34 + cells (3×10 2 to 5×10 2 cells/plate) were counted following plating in semisolid methylcellulose media. Colony formation was evaluated by determination of colony counts as a percentage of the corresponding control. The results are the means ± SD of three independent experiments. * P <0.01 compared with untreated control cells. RT-PCR results representative of three independent experiments are shown. GAPDH mRNA expression is shown as an internal control. The ALDH hi /CD34 + cells whose IER5 mRNA expression was analyzed by quantitative RT-PCR were derived from an AML patient (M1). The levels of the quantified RT-PCR products were normalized t o GAPDH expression in the same sample and were then expressed relative to the mRNA level of a normal control which was assigned a value of 1. D and E. Cell cycle analysis (D) and changes of mitochondrial membrane potential (ΔΨm) (E) in IER5 overexpressed or TMPP treated AML-derived ALDH hi /CD34 + cells. ALDH hi /CD34 + cells were purified from an AML patient (M1), and were then transfected with IER5 cDNA or were treated with TMPP (5 µM). The IER5-transfected or TMPP-treated cells were harvested after 3 days. The cell cycle distribution and the ΔΨm of the ALDH hi /CD34 + cells was analyzed using flow cytometric analysis. The FACS results are representative of three independent experiments. NC; Negative control.

Article Snippet: Full-length cDNAs encoding human IER5 and Cdc25B were obtained by RT-PCR using human bone marrow cDNA (BD Biosciences Clontech, Palo Alto, CA) as a template and were cloned into the eukaryotic expression vector pcDNA3.1/V5-His (Invitrogen, Carlsbad, CA).

Techniques: Selection, Negative Control, Staining, Purification, Cell Culture, Transfection, Microscopy, Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Derivative Assay, Cell Cycle Assay

Journal: PLoS ONE

Article Title: Transcriptional Repression of Cdc25B by IER5 Inhibits the Proliferation of Leukemic Progenitor Cells through NF-YB and p300 in Acute Myeloid Leukemia

doi: 10.1371/journal.pone.0028011

Figure Lengend Snippet: A. ALDH hi /CD34 + cells were purified from an AML patient (M1) and were cultured in semisolid methylcellulose media. The ALDH hi /CD34 + cells were either untransfected or were transfected with IER5 cDNA or with shRNA-#1, or -#2. After 14 days culture with or without TMPP (5 µM), the cells were then analyzed for their colony forming ability. Colony formation of these ALDH hi /CD34 + cells (3×10 2 to 5×10 2 cells/plate) was assessed after plating in semisolid methylcellulose media. B. Analysis of the mRNA expression of IER5 and Cdc25B in each colony using QRT-PCR and RT-PCR. C. The cells were viewed using phase-contrast microscopy after 14 days culture with or without TMPP (5 µM). Original magnification ×4. D. ALDH hi /CD34 + cells were purified from an AML patient (M1) and were cultured in semisolid methylcellulose media. The ALDH hi /CD34 + cells were either untransfected or were transfected with IER5 cDNA or Cdc25B cDNA. After 14 days culture with or without TMPP (5 µM), the cells were then analyzed for their colony forming ability. Colony formation of these ALDH hi /CD34 + cells (3×10 2 to 5×10 2 cells/plate) was assessed after plating in semisolid methylcellulose media. Colony formation was evaluated as a percentage of the corresponding control. E. Analysis of the mRNA expression of IER5 and Cdc25B in each colony using QRT-PCR and RT-PCR. The levels of the QRT-PCR products were normalized t o GAPDH expression in the same sample and were then expressed relative to the mRNA level of a normal control which was assigned a value of 1. RT-PCR results representative of three independent experiments are shown. GAPDH mRNA expression is shown as an internal control. The results are the means ± SD of three independent experiments. * P <0.01 compared with untreated control cells. F. The cells transfected with Cdc25B or IER5 cDNA were viewed using phase-contrast microscopy after 14 days culture with or without TMPP (5 µM). Original magnification ×4.

Article Snippet: Full-length cDNAs encoding human IER5 and Cdc25B were obtained by RT-PCR using human bone marrow cDNA (BD Biosciences Clontech, Palo Alto, CA) as a template and were cloned into the eukaryotic expression vector pcDNA3.1/V5-His (Invitrogen, Carlsbad, CA).

Techniques: Purification, Cell Culture, Transfection, shRNA, Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Microscopy

Journal: PLoS ONE

Article Title: FEZ2 Has Acquired Additional Protein Interaction Partners Relative to FEZ1: Functional and Evolutionary Implications

doi: 10.1371/journal.pone.0017426

Figure Lengend Snippet: A) Yeast two-hybrid screen performed with FEZ1 (221–392) as a bait using a human fetal brain cDNA library retrieved 16 proteins that were confirmed to interact all with both FEZ1(221–392) and FEZ2 (207–353) in the one-to-one assay. B) Yeast two-hybrid screen performed with FEZ2 (207–353) as a bait using human fetal brain and bone marrow cDNA libraries retrieved 59 proteins as preys. In the one-to-one confirmation, 19 of these proteins interacted only with FEZ2 (207–353) protein, 40 with both FEZ2 (207–353) and FEZ1 (221–392) and 36 interacted with all FEZ2 (207–353), FEZ1 (221–392) and UNC-76 (242–378).

Article Snippet: In this work, yeast two-hybrid screens of human fetal brain and bone marrow cDNA libraries (Clontech) were performed as described previously by using the yeast strain L40 (trp1-901, his3Δ200, leu2-3, ade2 LYS2::(lexAop)4-HIS3 URA3::(lexAop)8-lac GAL4) and human FEZ2 (207–353) as a bait in fusion with the LexA protein as encoded in the recombinant vector pBTM116 .

Techniques: Two Hybrid Screening, cDNA Library Assay