Journal: Leukemia

Article Title: IDH1 and IDH2 mutations in pediatric acute leukemia

doi: 10.1038/leu.2011.133

Figure Lengend Snippet: Genetic characteristics of the 515 pediatric leukemias analyzed

Article Snippet: 20 Site Directed Mutagenesis Human wildtype (WT) cDNA clones for IDH1 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_005896.2","term_id":"28178824","term_text":"NM_005896.2"}} NM_005896.2 ) and IDH2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_002168.2","term_id":"28178831","term_text":"NM_002168.2"}} NM_002168.2 ) in pCMV6 Entry vector were obtained from Origene (Origene, Rockville, MD).

Techniques:

Journal: Leukemia

Article Title: IDH1 and IDH2 mutations in pediatric acute leukemia

doi: 10.1038/leu.2011.133

Figure Lengend Snippet: Genomic structure of IDH1 and IDH2 and structural model of the location of R140 and R172 in the substrate-binding pocket of IDH2. (a) Illustration of the exon/intron structure of IDH1 and IDH2. The location within exon 4 of codon R132 in IDH1 and codon R140 in IDH2 are marked by arrows, and the surrounding nucleotide sequence and encoded amino acids are highlighted. Codon R132 in IDH1 and the homologous residue R172 in IDH2 are shown in red and codon 140 in IDH2 is shown in blue. (b) Model of the positions of the R140 and R172 amino acids in the IDH2 substrate binding pocket. The two protomers in the IDH2 homodimer are illustrated in green and purple, with the nitrogen atoms in amino acids R140 and R172 shown in blue. The bound substrate, isocitrate, is depicted with carbon atoms as yellow sticks and oxygen atoms in red. The manganese ion is shown as a grey sphere. Salt-bridges and hydrogen bonds are shown as dashed lines.

Article Snippet: 20 Site Directed Mutagenesis Human wildtype (WT) cDNA clones for IDH1 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_005896.2","term_id":"28178824","term_text":"NM_005896.2"}} NM_005896.2 ) and IDH2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_002168.2","term_id":"28178831","term_text":"NM_002168.2"}} NM_002168.2 ) in pCMV6 Entry vector were obtained from Origene (Origene, Rockville, MD).

Techniques: Binding Assay, Sequencing

Journal: Leukemia

Article Title: IDH1 and IDH2 mutations in pediatric acute leukemia

doi: 10.1038/leu.2011.133

Figure Lengend Snippet: Genetic characteristics of the AMLs with IDH1 / IDH2 mutations

Article Snippet: 20 Site Directed Mutagenesis Human wildtype (WT) cDNA clones for IDH1 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_005896.2","term_id":"28178824","term_text":"NM_005896.2"}} NM_005896.2 ) and IDH2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_002168.2","term_id":"28178831","term_text":"NM_002168.2"}} NM_002168.2 ) in pCMV6 Entry vector were obtained from Origene (Origene, Rockville, MD).

Techniques: Mutagenesis

Journal: Leukemia

Article Title: IDH1 and IDH2 mutations in pediatric acute leukemia

doi: 10.1038/leu.2011.133

Figure Lengend Snippet: Enzymatic analysis of the IDH1 and IDH2 mutant proteins. The activity of recombinant IDH1 and IDH2 proteins to catalyze the NADP+-dependent oxidative decarboxylation of isocitrate to α-KG using 30uM Isocitrate and 100uM NADP are shown in panels a and b, respectively, and their ability to catalyze the NADPH-dependent reduction of α–KG to 2-HG using 0.5 mM α-KG and 100uM NADPH are shown in c and d, respectively. All graphs are based on triplicate measurements with the mean ± standard deviations expressed as a relative level compared to WT:WT homodimers, with the latter set as 100%. The SD in panel c and d are < 0.03 and are thus below the resolution of the figure. (e) The intracellular level of 2-HG was measured by liquid chromatography/mass spectrometry in pediatric AML cells from primary diagnostic bone marrow samples. The data for mutant IDH1/IDH2 includes two leukemia samples containing IDH1 mutations (one with R132H and one with R132C, ▴), and two containing the R140Q IDH2 mutations (◆). The wild-type IDH1/IDH2 data was generated using two pediatric AML samples that lacked mutations in either IDH1 or IDH2 (●).

Article Snippet: 20 Site Directed Mutagenesis Human wildtype (WT) cDNA clones for IDH1 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_005896.2","term_id":"28178824","term_text":"NM_005896.2"}} NM_005896.2 ) and IDH2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_002168.2","term_id":"28178831","term_text":"NM_002168.2"}} NM_002168.2 ) in pCMV6 Entry vector were obtained from Origene (Origene, Rockville, MD).

Techniques: Mutagenesis, Activity Assay, Recombinant, Liquid Chromatography, Mass Spectrometry, Diagnostic Assay, Generated

Journal: PloS one

Article Title: Evidence for SH2 domain-containing 5'-inositol phosphatase-2 (SHIP2) contributing to a lymphatic dysfunction.

doi: 10.1371/journal.pone.0112548

Figure Lengend Snippet: Figure 2. Identification of T180A SHIP2 mutation in familial lymphedema. (A) Pedigree of the nucleus and extended family showing affected (filled) and unaffected (open) subjects with lymphedema, phenotyped by NIRF imaging and WES analysis revealing subjects who harbor T180A SHIP2, G315V HGF and N463S MAP3K7 mutations. NIRF imaging reveals (B) dermal backflow in medial left ankle of Subject #6 (bilateral lymphedema praecox) and also has abnormal lymphatic capillaries on thigh (not shown). (C) Abnormal lymphatic capillaries radiating from the injection site on the medial left ankle of Subject #9 which were also observed capillaries on the thigh on this unaffected subject. (D) Tortuous lymphatics draining the medial left ankle and lymphatic capillaries radiating from the left medial calf in Subject #12 (unaffected). All three subjects also appeared to have dilated lymphatics. doi:10.1371/journal.pone.0112548.g002

Article Snippet: Site-directed mutagenesis Human INPPL1 TrueORF cDNA Myc-DDK clone was obtained from OriGene.

Techniques: Mutagenesis, Imaging, Injection

Journal: PloS one

Article Title: Evidence for SH2 domain-containing 5'-inositol phosphatase-2 (SHIP2) contributing to a lymphatic dysfunction.

doi: 10.1371/journal.pone.0112548

Figure Lengend Snippet: Figure 3. Dysregulated HGF- and VEGFC-induced activation of AKT and ERK1/2 in SHIP2-deficient LEC. HDLEC (A,C,E,G) and TIME (B,D,F,H) were subjected to 48 hr SHIP2 siRNA and stimulated with HGF (A–D) or VEGFC (E–H) for the indicated times. Activation of AKT and ERK was determined by fluorescent double staining Western blotting of cell lysates with both phosphospecific antibodies and antibodies to total proteins. (See Fig S4 for representative blot images) Quantification of AKT (A,B, E,F) and ERK (C,D,G,H) activation in LEC and TIME by average mean fluorescence intensity (MFI) and represented as a ratio of pAKT-S473 to total AKT and pERK1/2 to total ERK1/2, respectively. Data presented as mean6SEM of MFI from 4 independent experiments. doi:10.1371/journal.pone.0112548.g003

Article Snippet: Site-directed mutagenesis Human INPPL1 TrueORF cDNA Myc-DDK clone was obtained from OriGene.

Techniques: Activation Assay, Double Staining, Western Blot, Fluorescence

Journal: PloS one

Article Title: Evidence for SH2 domain-containing 5'-inositol phosphatase-2 (SHIP2) contributing to a lymphatic dysfunction.

doi: 10.1371/journal.pone.0112548

Figure Lengend Snippet: Figure 4. SHIP2 is required for in vitro lymphangiogenesis in primary HDLEC. (A) MTS cell proliferation assay in response to growth factor stimulation for 48 hrs, normalized to 1% FBS of non-targeting (NT) siRNA (N = 3). (B) Cell adhesion assay of siRNA-transfected HDLEC onto BSA, collagen and fibronectin and quantification presented as area fraction of whole images (triplicates per experiment; 5 independent experiments). (C)

Article Snippet: Site-directed mutagenesis Human INPPL1 TrueORF cDNA Myc-DDK clone was obtained from OriGene.

Techniques: In Vitro, Proliferation Assay, Cell Adhesion Assay, Transfection