Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Role of Isocitrate Dehydrogenase 2 on DNA Hydroxymethylation in Human Airway Smooth Muscle Cells

doi: 10.1165/rcmb.2019-0323OC

Figure Lengend Snippet: Differences in global DNA 5hmC (5-hydroxymethylcytosine) levels, TET (ten-eleven translocation) activity, α-ketoglutarate (α-KG) levels, and mRNA levels of IDH2 (isocitrate dehydrogenase 2) between nonasthmatic and asthmatic airway smooth muscle (ASM) cells. (A and B) Global DNA 5hmC levels (A) and TET activity (B) in asthmatic ASM cells relative to nonasthmatic cells were assayed by ELISA. (C) α-KG levels in cells were assayed by a coupled enzyme assay. (D) mRNA levels of IDH2 were assayed by quantitative PCR (qPCR). Each data point represents an individual lung donor. Values are mean ± SEM (six lung donors without asthma and six with asthma). *P < 0.05, **P < 0.01, and ***P < 0.001 in comparison with nonasthmatic ASM cells.

Article Snippet: Alternatively, the cells were transfected with 500 ng of IDH2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_002168","term_id":"1780222522","term_text":"NM_002168"}} NM_002168 ) DNA plasmid (pCMV6-AC_IDH2, ORIGENE SC319226) or an empty vector (pCMV6-AC, ORIGENE PS100020) for IDH2 overexpression.

Techniques: Translocation Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Enzymatic Assay, Real-time Polymerase Chain Reaction

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Role of Isocitrate Dehydrogenase 2 on DNA Hydroxymethylation in Human Airway Smooth Muscle Cells

doi: 10.1165/rcmb.2019-0323OC

Figure Lengend Snippet: Knockdown of IDH2 by siIDH2 (siRNA against IDH2) decreased gene expression of IDH2, α-KG levels, TET activity, and global 5hmC levels in human asthmatic ASM cells. (A) mRNA levels of IDH2 were assayed by qPCR. (B) α-KG levels in cells were assayed by a coupled enzyme assay. (C and D) TET activity (C) and global 5hmC levels (D) were examined by ELISA. Each data point represents an individual lung donor. (A–C) All data are represented as the percentage change relative to lipofectamine (LF) controls and shown as mean values ± SEM (six lung donors with asthma). (D) Absolute amount of 5hmC in genomic DNA are present. *P < 0.05 and ****P < 0.0001 in comparison with siCTL (scramble siRNA control)-treated asthmatic ASM cells.

Article Snippet: Alternatively, the cells were transfected with 500 ng of IDH2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_002168","term_id":"1780222522","term_text":"NM_002168"}} NM_002168 ) DNA plasmid (pCMV6-AC_IDH2, ORIGENE SC319226) or an empty vector (pCMV6-AC, ORIGENE PS100020) for IDH2 overexpression.

Techniques: Expressing, Activity Assay, Enzymatic Assay, Enzyme-linked Immunosorbent Assay

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Role of Isocitrate Dehydrogenase 2 on DNA Hydroxymethylation in Human Airway Smooth Muscle Cells

doi: 10.1165/rcmb.2019-0323OC

Figure Lengend Snippet: Knockdown of IDH2 by siIDH2 reduced TGFB2 (transforming growth factor-β2) mRNA levels and hydroxymethylation of the TGFB2 promoter in human asthmatic ASM cells. (A) mRNA levels of TGFB2 in samples were assayed by qPCR. (B) Schematic diagram of CpG dinucleotide (GC) content (percentage) in the 5′ promoter region of TGFB2. In silico analysis identified the CpG islands (shaded in gray in the genomic DNA sequence) based on a GC content > 60% with an observed/expected ratio of 0.6 (MethPrimer). The PCR amplicon generated by PCR is indicated by the regions bounded by arrows. (C) Average of TGFB2 promoter methylation in nonasthmatic and asthmatic ASM cells was assayed by bisulfite sequencing. (D) mRNA levels of TGFB2 in asthmatic ASM cells after siRNA treatment were assayed by qPCR. (E and F) The average percentage of methylation (E) and hydroxymethylation (F) of each CpG site of the TGFB2 promoter in asthmatic ASM cells (six donors) was assayed by oxidative bisulfite sequencing. Four to six individual clones from each donor were picked for sequencing. Each data point represents the mean of six donors in LF controls (solid circles) and siCTL-treated (solid squares) and siIDH2-treated (open triangles) asthmatic ASM cells. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with LF controls. #P = 0.05 compared with siCTL-transfected cells. ATG = translation start site; Ave. = average; TSS = transcription start site; UTR = untranslated region.

Article Snippet: Alternatively, the cells were transfected with 500 ng of IDH2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_002168","term_id":"1780222522","term_text":"NM_002168"}} NM_002168 ) DNA plasmid (pCMV6-AC_IDH2, ORIGENE SC319226) or an empty vector (pCMV6-AC, ORIGENE PS100020) for IDH2 overexpression.

Techniques: In Silico, Sequencing, Amplification, Generated, Methylation, Methylation Sequencing, Oxidative Bisulfite Sequencing, Clone Assay, Transfection

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Role of Isocitrate Dehydrogenase 2 on DNA Hydroxymethylation in Human Airway Smooth Muscle Cells

doi: 10.1165/rcmb.2019-0323OC

Figure Lengend Snippet: Overexpression of IDH2 showed increased α-KG levels and TET activity in human nonasthmatic ASM cells. (A) mRNA levels of IDH2 were assayed by qPCR. (B) α-KG levels in cells were assayed by a coupled enzyme assay. (C and D) TET activity (C) and global 5hmC levels (D) were examined by ELISA. Each data point represents an individual lung donor. All data are represented as the percentage change relative to LF controls and shown as mean values ± SEM (four lung donors without asthma). *P < 0.05 compared with pCMV6-transfected ASM cells.

Article Snippet: Alternatively, the cells were transfected with 500 ng of IDH2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_002168","term_id":"1780222522","term_text":"NM_002168"}} NM_002168 ) DNA plasmid (pCMV6-AC_IDH2, ORIGENE SC319226) or an empty vector (pCMV6-AC, ORIGENE PS100020) for IDH2 overexpression.

Techniques: Over Expression, Activity Assay, Enzymatic Assay, Enzyme-linked Immunosorbent Assay, Transfection

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Role of Isocitrate Dehydrogenase 2 on DNA Hydroxymethylation in Human Airway Smooth Muscle Cells

doi: 10.1165/rcmb.2019-0323OC

Figure Lengend Snippet: Overexpression of IDH2 Altered Airway Smooth Muscle Phenotypic Gene Expression in Human Nonasthmatic Airway Smooth Muscle Cells

Article Snippet: Alternatively, the cells were transfected with 500 ng of IDH2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_002168","term_id":"1780222522","term_text":"NM_002168"}} NM_002168 ) DNA plasmid (pCMV6-AC_IDH2, ORIGENE SC319226) or an empty vector (pCMV6-AC, ORIGENE PS100020) for IDH2 overexpression.

Techniques: Over Expression, Expressing

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Role of Isocitrate Dehydrogenase 2 on DNA Hydroxymethylation in Human Airway Smooth Muscle Cells

doi: 10.1165/rcmb.2019-0323OC

Figure Lengend Snippet: Overexpression of IDH2 Altered 5hmC Enrichment at the Gene Promoter of COL3A , SMAD3 , and TGFB2

Article Snippet: Alternatively, the cells were transfected with 500 ng of IDH2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_002168","term_id":"1780222522","term_text":"NM_002168"}} NM_002168 ) DNA plasmid (pCMV6-AC_IDH2, ORIGENE SC319226) or an empty vector (pCMV6-AC, ORIGENE PS100020) for IDH2 overexpression.

Techniques: Over Expression

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 ORF clone , Nucleic acid , OriGene , RC201152 , Replaces Invitrogen plasmid in pOTB7.

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

Journal: eLife

Article Title: Registered report: The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate

doi: 10.7554/eLife.12626

Figure Lengend Snippet:

Article Snippet: IDH2 R172K ORF in pCMV6 , Plasmid , Origene , RC400103 , .

Techniques: Modification, Plasmid Preparation, Protein Extraction, Protease Inhibitor, Spectrophotometry, DC Protein Assay, Staining

Journal: The Journal of Biological Chemistry

Article Title: Impact of structural biologists and the Protein Data Bank on small-molecule drug discovery and development

doi: 10.1016/j.jbc.2021.100559

Figure Lengend Snippet: A , allosteric inhibitor enasidenib ( red ) targeting homodimeric IDH2 ( yellow and blue monomers ; PDB ID: 5i96 ). B , superposition of apo ( green PDB ID: 5i95 ) and inhibited ( yellow and blue monomers ; PDB ID: 5i96 ) forms of IDH1 computed by overlaying the central domains (boxed in red ). Arrows indicate the locations of the two active sites. The view in ( B ) is rotated ∼45° about the vertical relative to ( A ).

Article Snippet: In 2018, the PDB archive housed six MX structures of human IDH2, all of which were contributed by biopharmaceutical companies ( i.e. , Novartis or Agios).

Techniques: