Journal: Nature

Article Title: Mutant IDH inhibits HNF-4α to block hepatocyte differentiation and promote biliary cancer.

doi: 10.1038/nature13441

Figure Lengend Snippet: Figure 2 | Mutant IDH blocks hepatocyte differentiation by silencing HNF- 4a. a, Heat map of GSEA showing top-ranked gene sets distinguishing IDH1(R132C) or IDH2(R172K) from wild-type (WT) or empty vector (EV) control hepatoblasts (pairwise analysis; replicates for each condition; see Methods). NES, normalized enrichment score; NS, not significant. b, c, Hepatoblasts analysed by immunoblot (b) and qRT–PCR (c). d, e, Analysis of wild-type hepatoblasts expressing the indicated short hairpin (sh)RNAs (uncoated plates). CTL, control. d, Hepatocyte sphere formation. e, Proliferation of shRNA-expressing hepatoblast cells co-expressing EV or shRNA-resistant Hnf4a(1) complementary DNA. f–h, Control and R132C- expressing hepatoblasts co-expressing vector control (EV2) or HNF-4a, grown on uncoated plates. f, Hepatocyte sphere formation. g, Hepatocyte gene expression. h, Proliferation. Scale bars, 100mm (d), 250mm (f). *P , 0.05.

Article Snippet: The R140Q and R172K mutations were introduced into human IDH2 cDNA (Origene, MD # SC319226, NCBI RefSeq accession number NM_002168.3) using the Quickchange kit (Agilent Lexington, MA #200521) as per the manufacturer’s instructions.

Techniques: Mutagenesis, Plasmid Preparation, Control, Western Blot, Quantitative RT-PCR, Expressing, shRNA, Gene Expression

Journal: Nature Communications

Article Title: Age-impaired remyelination is associated with dysregulated microglial transitions

doi: 10.1038/s41467-025-64906-w

Figure Lengend Snippet: a Representative scatterplot showing Ly6g + neutrophils, Ly6c + monocytes and Ly6c - Ly6g - microglia from Cd45 + Mrc1 - Cd11b + cells. b Unsupervised clustering of 47,871 microglia and the ( c ) proportional contribution of each cluster isolated from d – f young, 2-month-old and g – i middle-aged, 10-month-old male mice from naïve (n young = 10, n middle-aged = 10), 7 DPI (n young = 8, n middle-aged = 12) and 21 DPI (n young = 10, n middle-aged = 12) conditions, respectively. j UMAP depicting the expression of Idh2—a protein involved in the oxidative phosphorylation pathway. k Proportions of each sample (naïve (n young = 10, n middle-aged = 10), 7 DPI (n young = 8, n middle-aged = 12) and 21 DPI (n young = 10, n middle-aged = 12)) present in neutrophil and monocyte populations. Two-way analysis of variance (ANOVA) with Sidak’s post-hoc test. Each point represents two animals combined, and all data are presented as mean ± SEM. Metabolic pathway activity across ( l ) microglial states identified in (Fig. ), throughout remyelination in ( m ) young mice and ( n ) young combined with middle-aged mice as a function of the genes found in the KEGG metabolic pathways. Source data are provided as a source data file.

Article Snippet: The pellet was resuspended for 40 min in 200 μL of intracellular stain cocktail containing HBSS, 10 μL of BD Brilliant Stain Buffer (BD Biosciences, Cat. No. 566385) and the following antibodies at a 1:100 dilution: Alexa Fluor 647 GAPDH Antibody (#3907, Cell Signaling Technology, Cat. No. 3907S), Alexa Fluor 700 Ki-67 Antibody (Clone 16A8, BioLegend, Cat. No. 652420), Alexa Fluor 488 IRF7 Antibody (Bioss, Cat. No. BS-3196R-A488), APC-eFluor 780 CD68 Antibody, (FA-11, eBioscience, Cat. No. 47-0681-82), DyLight 350 IGF-I/IGF-1 Antibody (Novus Biologicals, Cat. No. NBP2-48001UV), DyLight 594 IDH2 Antibody (Novus Biologicals, Cat. No. NBP2-22166DL594), Brilliant Violet 711TM CD206 Antibody (Clone C068C2, BioLegend, Cat. No. 141727), PE Osteopontin Antibody (R&D Systems, Cat. No. IC808P), PE/Cyanine7 Phospho STAT1 Antibody (Clone A15158B, BioLegend, Cat. No. 686408), PerCP-eFluorTM 710 Ccl3 Antibody (eBioscience, Cat. No. 46-7532-82) and centrifuged (600 × g , 5 min, 4 °C).

Techniques: Isolation, Expressing, Phospho-proteomics, Activity Assay

Journal: Nature communications

Article Title: Yap regulates skeletal muscle fatty acid oxidation and adiposity in metabolic disease.

doi: 10.1038/s41467-021-23240-7

Figure Lengend Snippet: Fig. 4 Idh2 upregulation prevents impaired fatty acid oxidation associated with Yap knockdown. a Graph showing the changes in chromatin accessibility and transcript abundance at enhancers or promoters (TSS). Numbers within bars indicate the number of genes detected in each comparison (FDR < 0.05, rank-MANOVA), (b) sequencing tracks of Idh2 transcript in AAV6:lacZ-shRNA (blue) and AAV:Yap shRNA (red). Each track represent a biological replicate from an individual animal (n = 3), (c) incomplete fatty acid oxidation in EDL muscles analysed 2 weeks after injection of control, AAV6:Yap- shRNA, AAV6:Idh2 vectors or both (n = 10 biologically independent animals, mean ± SEM, * shows significant difference, one-way ANOVA with Tukey’s multiple comparison test with exact p values provided in source data), (d) representative western blots of Yap, Idh2, and total protein levels in TA muscles from mice treated as in (c) (n = 5 biologically independent animals), (e) Representative H and E images of TA muscle from mice treated as in (c) (n = 5 biologically independent animals, scale bars indicate 50 µm).

Article Snippet: Primary antibodies used were total YAP (#14074) and total IDH2 (#56439); all 1/1000 dilution from Cell Signalling Technologies and GAPDH (#SC-32233); 1/20,000 dilution from Santa-Cruz Biotechnology.

Techniques: Knockdown, Comparison, Sequencing, shRNA, Muscles, Injection, Control, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: Cancer-associated metabolite 2-hydroxyglutarate accumulates in acute myelogenous leukemia with isocitrate dehydrogenase 1 and 2 mutations

doi: 10.1084/jem.20092506

Figure Lengend Snippet: Identification of 13 AML patients bearing an IDH1 R132 or IDH2 R172 mutation

Article Snippet: The human IDH1 cDNA (GenBank/EMBL/DDBJ accession no. NM_005896 ) and IDH2 cDNA (GenBank/EMBL/DDBJ accession no. NM_002168 ) were purchased from OriGene Technologies.

Techniques:

Journal: The Journal of Experimental Medicine

Article Title: Cancer-associated metabolite 2-hydroxyglutarate accumulates in acute myelogenous leukemia with isocitrate dehydrogenase 1 and 2 mutations

doi: 10.1084/jem.20092506

Figure Lengend Snippet: IDH1/2 mutant AML cells and sera have increased levels of 2-HG. (A) Extracts from IDH1/2 WT ( n = 10) and IDH1/2 mutant ( n = 16) patient leukemia cells obtained at presentation and relapse, and IDH1 R132 mutant leukemia cells grown in culture for 14 d ( n = 14) were analyzed by LC-MS to measure levels of 2-HG. (B) 2-HG was measured in sera of patients with IDH1 WT or IDH1 R132 mutant leukemia. In A and B, each point represents an individual patient sample. Diamonds represent WT, circles represent IDH1 mutants, and triangles represent IDH2 mutants. Horizontal bars indicate the mean. * indicates a statistically significant difference relative to WT patient cells (P < 0.05).

Article Snippet: The human IDH1 cDNA (GenBank/EMBL/DDBJ accession no. NM_005896 ) and IDH2 cDNA (GenBank/EMBL/DDBJ accession no. NM_002168 ) were purchased from OriGene Technologies.

Techniques: Mutagenesis, Liquid Chromatography with Mass Spectroscopy

Journal: The Journal of Experimental Medicine

Article Title: Cancer-associated metabolite 2-hydroxyglutarate accumulates in acute myelogenous leukemia with isocitrate dehydrogenase 1 and 2 mutations

doi: 10.1084/jem.20092506

Figure Lengend Snippet: IDH1/2 mutant AML cells do not display altered levels of central carbon metabolites. Extracts from leukemia cells of AML patients carrying an IDH1/2 mutant allele (mutant; n = 16) or WT ( n = 10) obtained at initial presentation and relapse were assayed by LC-MS for levels of α-KG, succinate, malate, and fumarate. Each point represents an individual patient sample. Open circles represent WT, closed circles represent IDH1 mutants, and triangles represent IDH2 mutants. Horizontal bars represent the mean. There were no statistically significant differences between the WT and IDH1/2 mutant AML samples.

Article Snippet: The human IDH1 cDNA (GenBank/EMBL/DDBJ accession no. NM_005896 ) and IDH2 cDNA (GenBank/EMBL/DDBJ accession no. NM_002168 ) were purchased from OriGene Technologies.

Techniques: Mutagenesis, Liquid Chromatography with Mass Spectroscopy

Journal: The Journal of Experimental Medicine

Article Title: Cancer-associated metabolite 2-hydroxyglutarate accumulates in acute myelogenous leukemia with isocitrate dehydrogenase 1 and 2 mutations

doi: 10.1084/jem.20092506

Figure Lengend Snippet: Recombinant IDH1 R132C and IDH2 R172K produce 2-HG. (A) LC-MS analysis of in vitro reactions using recombinant IDH1 R132C and IDH2 R172K confirms that 2-HG and not isocitrate is the end product of the mutant enzyme reactions. Reactions were performed in triplicate in each of two independent experiments; typical chromatograms are presented. (B) The WT IDH1 enzyme catalyzes the oxidative decarboxylation of isocitrate to α-KG, with the concomitant reduction of NADP to NADPH. The IDH1 R132C and IDH2 R172K mutants reduce α-KG to 2-HG while oxidizing NADPH to NADP. These are referred to in the text as the “forward” and “partial reverse” reactions, respectively.

Article Snippet: The human IDH1 cDNA (GenBank/EMBL/DDBJ accession no. NM_005896 ) and IDH2 cDNA (GenBank/EMBL/DDBJ accession no. NM_002168 ) were purchased from OriGene Technologies.

Techniques: Recombinant, Liquid Chromatography with Mass Spectroscopy, In Vitro, Mutagenesis

Journal:

Article Title: Hippocampal responsiveness to 17?-estradiol and equol after long-term ovariectomy: Implication for a therapeutic window of opportunity

doi: 10.1016/j.brainres.2011.01.029

Figure Lengend Snippet: Western blot analysis of IDH2 (A) and SDHα (B) expression was performed on NHP hippocampal samples from both of the hormone treatment paradigms as well as controls. Expression levels for each sample were normalized to beta-actin levels. Expression levels were then normalized to the control animals (controls were set to 100%). Statistically significant differences were calculated using a two-tailed, one-way analysis of variance (ANOVA) followed by a Bonferroni post-hoc correction. Hormone treatment significantly affected expression of SDHα in the NHP hippocampus. Bars represent % control ± S.E.M., n = 8 for each condition, **p<0.01.

Article Snippet: Isocitrate dehydrogenase 2 (IDH2) , 44 KDa , H00003418 , Novus Biologicals, Littleton, CO , 1/1000 , Mouse , Brinton Lab, unpublished data.

Techniques: Western Blot, Expressing, Control, Two Tailed Test

Journal:

Article Title: Hippocampal responsiveness to 17?-estradiol and equol after long-term ovariectomy: Implication for a therapeutic window of opportunity

doi: 10.1016/j.brainres.2011.01.029

Figure Lengend Snippet: List of antibodies used for Western blotting

Article Snippet: Isocitrate dehydrogenase 2 (IDH2) , 44 KDa , H00003418 , Novus Biologicals, Littleton, CO , 1/1000 , Mouse , Brinton Lab, unpublished data.

Techniques: Western Blot