Journal: eLife

Article Title: Registered report: IDH mutation impairs histone demethylation and results in a block to cell differentiation

doi: 10.7554/eLife.10860

Figure Lengend Snippet:

Article Snippet: IDH2 R172K ORF clone , Nucleic acid , OriGene , RC400103 , Original generated by authors.

Techniques: Bradford Assay, Reporter Assay, Cell Culture, Plasmid Preparation, Generated, Western Blot, Staining, Protease Inhibitor, Protein Concentration

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: 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: 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: 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: 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: Cancer research

Article Title: IDH2 Mutations Define a Unique Subtype of Breast Cancer with Altered Nuclear Polarity

doi: 10.1158/0008-5472.CAN-16-0298

Figure Lengend Snippet: (A) Non-synonymous somatic IDH2, TET2, PIK3CA, and PIK3R1 mutations identified in the 13 SPCRPs studied here by massively parallel sequencing (WES or MSK-IMPACT), SNaPshot or Sanger sequencing. (B) Mutation plot shows the domain structure of IDH2 and the non-synonymous IDH2 mutations identified in SPCRP and in common forms of breast cancer published by TCGA available from cBioPortal (28). (C) 2HG analysis in IDH2-mutant SPCRP (case 12) and IDH wild-type invasive ductal carcinoma of no special type (IDC). (D) IDH2/TET2-mutant SPCRPs display global DNA hypermethylation assessed by Infinium MethylationEPIC BeadChip as compared to IDH2/TET2 wild-type IDCs (left). Unsupervised hierarchical clustering using all methylation values/ genes of six IDH2/TET2-mutant SPCRPs and two IDH2/TET2 wild-type IDCs using complete linkage and Euclidean distance (right). The 1000 genes with the highest variance across samples are displayed. Methylation status is color coded according to the legend. (E) H3K27me3 immunohistochemical analysis of four IDH2-mutant SPCRPs (top row) and four IDH2 wild-type IDCs (bottom row). Nuclear immunoreactivity for H3K27me3 was quantified using the H-score. *, p<0.05; Mann-Whitney U test.

Article Snippet: DNA transfections and analysis of transgene expression The human IDH2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_002168","term_id":"588282795","term_text":"NM_002168"}} NM_002168 ) cDNA ORF clone pCMV6-IDH2::Myc-DDK was purchased from Origene (RC201152), and the R172S mutation introduced using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs) following the manufacturer’s recommendations.

Techniques: Sequencing, Mutagenesis, Methylation, Immunohistochemical staining, MANN-WHITNEY

Journal: Cancer research

Article Title: IDH2 Mutations Define a Unique Subtype of Breast Cancer with Altered Nuclear Polarity

doi: 10.1158/0008-5472.CAN-16-0298

Figure Lengend Snippet: (A) Detection of 2HG as a readout of IDH2WT and IDH2R172S enzymatic activity in total protein lysates (left) and in conditioned media (right) of MCF10AP and MCF10AH1047R cells expressing empty vector control, IDH2WT or IDH2R172S. The same cell lysates from MCF10AP and MCF10AH1047R cells were analyzed for IDH2 protein expression by western blotting. Total tubulin was used as loading control; ns: not significant; ***P<0.001; ****P<0.0001; error bars represent standard deviation of mean. (B) Whole cell lysates of MCF10AP and MCF10AH1047R cells were analyzed for IDH2, total and phosphorylated RB, E-cadherin, and tubulin protein expression by western blotting (left), and quantified using near-infrared detection (LI-COR; Odyssey) (right); error bars represent standard deviation of mean.

Article Snippet: DNA transfections and analysis of transgene expression The human IDH2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_002168","term_id":"588282795","term_text":"NM_002168"}} NM_002168 ) cDNA ORF clone pCMV6-IDH2::Myc-DDK was purchased from Origene (RC201152), and the R172S mutation introduced using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs) following the manufacturer’s recommendations.

Techniques: Activity Assay, Expressing, Plasmid Preparation, Western Blot, Standard Deviation

Journal: Frontiers in Cell and Developmental Biology

Article Title: LncRNA OIP5-AS1 Regulates the Warburg Effect Through miR-124-5p/IDH2/HIF-1α Pathway in Cervical Cancer

doi: 10.3389/fcell.2021.655018

Figure Lengend Snippet: Sequences of primers of qPCR, si-RNA, and miRNA.

Article Snippet: IDH2 , 1:1000 , 56439 , Cell Signaling Technology.

Techniques: Sequencing

Journal: Frontiers in Cell and Developmental Biology

Article Title: LncRNA OIP5-AS1 Regulates the Warburg Effect Through miR-124-5p/IDH2/HIF-1α Pathway in Cervical Cancer

doi: 10.3389/fcell.2021.655018

Figure Lengend Snippet: Antibody information.

Article Snippet: IDH2 , 1:1000 , 56439 , Cell Signaling Technology.

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: LncRNA OIP5-AS1 Regulates the Warburg Effect Through miR-124-5p/IDH2/HIF-1α Pathway in Cervical Cancer

doi: 10.3389/fcell.2021.655018

Figure Lengend Snippet: OIP5-AS1 mediates the promoting effect of hypoxia on Warburg effect is IDH2 dependent. (A) Levels of LDHA enzymatic activity (A) , representative of GLUT1 protein expression images (B) , and levels of GLUT1 and LDHA mRNA (C) in Hela cells knocking down OIP5-AS1 or its control under normoxic or hypoxic conditions for 24 h. Data were shown as mean ± SD of three individual experiments. ns was P > 0.05, *** was P < 0.001, and P value was calculated by Student’s t test. (D) We established a Hela cells overexpressing IDH2 (Over-IDH2) and its control cells (Plasmid), and used western blot to detect the expression of IDH2 protein in Over-IDH2 or Plasmid Hela cells after knocking down OIP5-AS1. (E,F) Levels of lactate (E) in the culture medium and intracellular glucose (F) of Hela cells after culturing for 24 h under hypoxia. Data were shown as mean ± SD of three individual experiments. *** was P < 0.001 and P value was calculated by post hoc comparisons.

Article Snippet: IDH2 , 1:1000 , 56439 , Cell Signaling Technology.

Techniques: Activity Assay, Expressing, Control, Plasmid Preparation, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: LncRNA OIP5-AS1 Regulates the Warburg Effect Through miR-124-5p/IDH2/HIF-1α Pathway in Cervical Cancer

doi: 10.3389/fcell.2021.655018

Figure Lengend Snippet: OIP5-AS1 regulates miR-124-5p expression by both directly targeting and Ago2-dependent manner, and miR-124-5p target inhibition of IDH2 expression in cervical cancer cell. (A) Predicted binding sites of miR-124-5p on OIP5-AS1 transcript (black) and IDH2 transcript (black). (B) RT-qPCR analysis of the indicated miR-124-5p levels in Hela, Caski, and Siha cells under normoxic or hypoxic conditions for 24 h. Data were shown as mean ± SD of three individual experiments. *** was P < 0.001 and P value was calculated by Student’s t test. (C,D) RT-qPCR analysis of the indicated miR-124-5p or OIP5-AS1 levels in Hela, Caski, and Siha cells over-expressing miR-124-5p or expressing knockdown miR-124-5p under normoxic or hypoxic conditions for 24 h. Data were shown as mean ± SD of three individual experiments. * was P < 0.05, ** was P < 0.01 and, *** was P < 0.001, and P value was calculated by Student’s t test. (E) miR-124-5p-NC, mimic and inhibitor was co-transferred with OIP5-AS1 wild type (WT) or mutant (MUT) transcript, and then detected luciferase activity. Data were shown as mean ± SD of three individual experiments. ns was P > 0.05 and *** was P < 0.001, and P value was calculated by post hoc comparisons. (F) RT-qPCR analysis of the indicated miR-124-5p and OIP5-AS1 levels associated with AGO2 after RIP assay in Hela cells. Data were shown as mean ± SD of three individual experiments. *** was P < 0.001, and P value was calculated by post hoc comparisons. (G,H) Dual-luciferase reporter assay shows that miR-124-5p targets inhibition of IDH2 expression (G) , and Western blot analysis shows that OIP5-AS1 promotes IDH2 expression via inhibiting miR-124-5p expression (H) . Data were shown as mean ± SD of three individual experiments. ns was P > 0.05 and *** was P < 0.001, and P value was calculated by post hoc comparisons in panel (G) and by Student’s t test in panel (H) . (I) Schematic illustration of the proposed model depicting OIP5-AS1 promotes IDH2 expression via inhibiting miR-124-5p expression.

Article Snippet: IDH2 , 1:1000 , 56439 , Cell Signaling Technology.

Techniques: Expressing, Inhibition, Binding Assay, Quantitative RT-PCR, Knockdown, Mutagenesis, Luciferase, Activity Assay, Reporter Assay, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: LncRNA OIP5-AS1 Regulates the Warburg Effect Through miR-124-5p/IDH2/HIF-1α Pathway in Cervical Cancer

doi: 10.3389/fcell.2021.655018

Figure Lengend Snippet: OIP5-AS1 regulates the Warburg effect through IDH2 is HIF-1α dependent. (A,B) Levels of IDH2, HIF-1α, GLUT1, and LDHA protein expression in Hela cells using western blot. (C–F) Levels of lactate (C) in the culture medium and intracellular glucose (D) of Hela cells after culturing for 24 h under hypoxia, and RT-qPCR indicated GLUT1/LDHA mRNA levels, western blot analysis showed IDH2, HIF-1α, GLUT1, and LDHA protein expression. Data were shown as mean ± SD of three individual experiments. P value was calculated by post hoc comparisons. *** P < 0.001.

Article Snippet: IDH2 , 1:1000 , 56439 , Cell Signaling Technology.

Techniques: Expressing, Western Blot, Quantitative RT-PCR