Journal: The Analyst

Article Title: A high-throughput SAMDI-mass spectrometry assay for isocitrate dehydrogenase 1

doi: 10.1039/d0an00174k

Figure Lengend Snippet: IDH1 catalyzes the conversion of isocitrate to αKG.

Article Snippet: The membrane was then blocked using Odyssey® TBS blocking buffer (LI-COR, Lincoln, NE) at room temperature for 1 hour and incubated overnight with rabbit IgG anti-IDH1 (Cell Signaling Technology, Danvers, MA) at a 1 : 1000 dilution and mouse IgG1 anti-HSP70 (BD Biosciences, Franklin Lanes, NJ) at a 1 : 2000 dilution at 4 °C.

Techniques:

Journal: The Analyst

Article Title: A high-throughput SAMDI-mass spectrometry assay for isocitrate dehydrogenase 1

doi: 10.1039/d0an00174k

Figure Lengend Snippet: (a) Scheme for the preparation and analysis of recombinant IDH1 samples. (b) αKG concentrations measured in the 384-sample biochemical screen (7 samples did not produce either 12C or 13C αKG capture).

Article Snippet: The membrane was then blocked using Odyssey® TBS blocking buffer (LI-COR, Lincoln, NE) at room temperature for 1 hour and incubated overnight with rabbit IgG anti-IDH1 (Cell Signaling Technology, Danvers, MA) at a 1 : 1000 dilution and mouse IgG1 anti-HSP70 (BD Biosciences, Franklin Lanes, NJ) at a 1 : 2000 dilution at 4 °C.

Techniques: Recombinant

Journal: The Analyst

Article Title: A high-throughput SAMDI-mass spectrometry assay for isocitrate dehydrogenase 1

doi: 10.1039/d0an00174k

Figure Lengend Snippet: IDH1 expression level as determined with western blot and αKG concentration as measured with SAMDI-MS for untreated cells and cells treated with transfection agent, control sequence siRNA and IDH1-targeting siRNA. All data are expressed as mean ± standard deviation with n = 2 experimental replicates.

Article Snippet: The membrane was then blocked using Odyssey® TBS blocking buffer (LI-COR, Lincoln, NE) at room temperature for 1 hour and incubated overnight with rabbit IgG anti-IDH1 (Cell Signaling Technology, Danvers, MA) at a 1 : 1000 dilution and mouse IgG1 anti-HSP70 (BD Biosciences, Franklin Lanes, NJ) at a 1 : 2000 dilution at 4 °C.

Techniques: Expressing, Western Blot, Concentration Assay, Transfection, Sequencing, Standard Deviation

Journal: Molecular cancer research : MCR

Article Title: Targeting IDH1 as a pro-senescent therapy in high-grade serous ovarian cancer

doi: 10.1158/1541-7786.MCR-18-1233

Figure Lengend Snippet: Ovcar3 cells were infected with lentivirus expressing two independent shRNAs targeting IDH1 or treated with 15μM GSK864 (IDH1i). Empty vector or DMSO was used as a control, respectively.

Article Snippet: The following antibodies were obtained from the indicated suppliers: rabbit anti-IDH1 (Cell Signaling), rabbit anti-Lamin B1 (Abcam), rabbit anti-Cyclin A (Abcam), mouse anti-PCNA (Cell Signaling), mouse anti-MCM3 (Santa Cruz Biotechnologies), mouse anti-Vinculin (Sigma-Aldrich), mouse anti-Beta Actin (Sigma-Aldrich), rat anti-BrdU (Abcam), mouse anti-PML (Santa Cruz Biotechnologies), mouse anti-γH2AX (EMD Millipore), rabbit anti-53BP1 (Bethyl), Fluorescein donkey anti-rat IgG (Jackson ImmunoResearch), Cy™3 donkey anti-mouse (Jackson ImmunoResearch).

Techniques: Infection, Expressing, Plasmid Preparation

Journal: Molecular cancer research : MCR

Article Title: Targeting IDH1 as a pro-senescent therapy in high-grade serous ovarian cancer

doi: 10.1158/1541-7786.MCR-18-1233

Figure Lengend Snippet: (A-F) Ovcar3 cells were infected with lentivirus expressing two independent short hairpin RNAs (shRNAs) targeting IDH1 (shIDH1). Empty vector was used as a control.

Article Snippet: The following antibodies were obtained from the indicated suppliers: rabbit anti-IDH1 (Cell Signaling), rabbit anti-Lamin B1 (Abcam), rabbit anti-Cyclin A (Abcam), mouse anti-PCNA (Cell Signaling), mouse anti-MCM3 (Santa Cruz Biotechnologies), mouse anti-Vinculin (Sigma-Aldrich), mouse anti-Beta Actin (Sigma-Aldrich), rat anti-BrdU (Abcam), mouse anti-PML (Santa Cruz Biotechnologies), mouse anti-γH2AX (EMD Millipore), rabbit anti-53BP1 (Bethyl), Fluorescein donkey anti-rat IgG (Jackson ImmunoResearch), Cy™3 donkey anti-mouse (Jackson ImmunoResearch).

Techniques: Infection, Expressing, Plasmid Preparation

Journal: Molecular cancer research : MCR

Article Title: Targeting IDH1 as a pro-senescent therapy in high-grade serous ovarian cancer

doi: 10.1158/1541-7786.MCR-18-1233

Figure Lengend Snippet: Ovcar3 and Ovcar10 cells were infected with two independent hairpins targeting IDH1. Empty vector was used as a control.

Article Snippet: The following antibodies were obtained from the indicated suppliers: rabbit anti-IDH1 (Cell Signaling), rabbit anti-Lamin B1 (Abcam), rabbit anti-Cyclin A (Abcam), mouse anti-PCNA (Cell Signaling), mouse anti-MCM3 (Santa Cruz Biotechnologies), mouse anti-Vinculin (Sigma-Aldrich), mouse anti-Beta Actin (Sigma-Aldrich), rat anti-BrdU (Abcam), mouse anti-PML (Santa Cruz Biotechnologies), mouse anti-γH2AX (EMD Millipore), rabbit anti-53BP1 (Bethyl), Fluorescein donkey anti-rat IgG (Jackson ImmunoResearch), Cy™3 donkey anti-mouse (Jackson ImmunoResearch).

Techniques: Infection, Plasmid Preparation

Journal: Cancer Science

Article Title: Pharmacological characterization of TQ 05310, a potent inhibitor of isocitrate dehydrogenase 2 R140Q and R172K mutants

doi: 10.1111/cas.14152

Figure Lengend Snippet: Genetic mutations in isocitrate dehydrogenase (IDH)1/2

Article Snippet: Monoclonal antibodies against IDH1 and Myc‐tag were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Mutagenesis

Journal: Cell Metabolism

Article Title: Polyamines and eIF5A Hypusination Modulate Mitochondrial Respiration and Macrophage Activation

doi: 10.1016/j.cmet.2019.05.003

Figure Lengend Snippet:

Article Snippet: Anti-IDH1 , Cell signalling , D2H1.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Construct, Plasmid Preparation, Sequencing, Clone Assay, Software

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Sensitive Quantitative Proteomics of Human Hematopoietic Stem and Progenitor Cells by Data-independent Acquisition Mass Spectrometry

doi: 10.1074/mcp.TIR119.001431

Figure Lengend Snippet: Proteome-transcriptome correlation in human hematopoietic stem and progenitor cell subpopulations. ( A ) Nonsupervised hierarchical clustering (Euclidean distance) heatmap of the intensity for the transcripts identified in HSCs/MPPs (referred to as HSCs), CMPs, GMPs, and MEPs (shades of red) isolated from five different HSPC donors (shades of blue). The transcript intensities are centered and scaled and depicted in color shades from red to blue. The transcripts with missing transcript intensity values in all samples were removed because they could not be handled by the clustering algorithm. Remaining missing transcript intensity values are shown in white. Clustering was observed mostly according to cell type, not according to donor. ( B ) GO enrichment analysis showed good alignment of protein and mRNA data. GSEA was performed for ranked mRNA and protein lists using GO processes from the Gene Ontology Consortium database as gene sets. Shown are normalized enrichment scores for the individual gene sets. Significantly up-regulated gene sets are marked in blue color; significantly downregulated gene sets are marked in red color. Significance was defined as FDR < 0.25, specific cell subpopulations were compared with the average of the remaining three cell types, and log2(fold change) was used as ranking criterion. Empty fields mean that no enrichment could be calculated. Abbreviations: MEGA, megakaryocyte; MAPK, mitogen-activated protein kinase; PI3K, phosphoinositide-3-kinase; PLC, phospholipase C. ( C ) Correlation between proteomics and transcriptomics data for HSCs/MPPs (referred to as HSCs), CMPs, GMPs, and MEPs. Dots are depicted in red when the FDR was below 0.01 both for protein and RNA data, orange when FDR < 0.01 for protein data, purple when FDR < 0.01 for RNA data, and gray when FDR ≥ 0.01 for both protein and RNA data. ( D ) Network analysis of significantly up-regulated proteins with concomitant significantly downregulated mRNA in HSCs/MPPs. Two clusters were especially prominent, including the snoRNPs and telomerase complex proteins GAR1, DKC1, NOP10, NHP2, and the quiescence-inducing NAD(P)H-producing proteins IDH1, IDH3A, and IDH3B. HSCs/MPPs were compared with the average of the other three subpopulations; cutoffs were set at FDR < 0.01 for protein and RNA data. Colors depict GO terms found enriched in B .

Article Snippet: For FACS analysis of intracellular isocitrate dehydrogenase 1 (IDH1) expression, the IntraPrep Leukocytic Permeabilization Reagent Kit (Beckman Coulter, Brea, CA) was used together with phycoerythrin (PE)-conjugated anti-IDH1, D2H1 (Cell Signaling Technology, Danvers, MA) and PE-conjugated isotype control, DA1E (Cell Signaling Technology).

Techniques: Isolation

Journal: Aging (Albany NY)

Article Title: An expression screen for aged-dependent microRNAs identifies miR-30a as a key regulator of aging features in human epidermis

doi: 10.18632/aging.101326

Figure Lengend Snippet: ( A ) A list of genes potentially targeted by miR-30a according to transcriptome data available in the GEO database. The number of conserved miR-30a MRE is indicated for each gene. The potential expression of these genes in human epidermis (according to the Human Protein Atlas database) is indicated. Five genes exhibiting a least one miR-30a conserved MRE and expressed in human epidermis, were analyzed by QPCR in RNA samples from skin equivalent (SE) model mimicking aging. The expression profile is indicated for these 5 genes. ( B ) The expression levels of AVEN , IDH1 and LOX were evaluated by QPCR in RNA samples from SEs mimicking aging with a long time of culture from day 35 (D35) to day 100 (D100). Results are mean +/− SD from three independent samples. *P< 0,05, ****P<0,0001. ( C ) Expression of AVEN, IDH1 or LOX in young or old human skin sections assessed by immunofluorescence for AVEN and IDH1 and by immunohistochemistry for LOX. (D) Quantification of the AVEN, IDH1 and LOX labeled area in the epidermis from young or old human skin sections. Results are mean +/− SD from three independent samples. ***P<0,001, **P<0,01.

Article Snippet: The membrane was incubated overnight at 4°C in TBS-T with primary antibody specific to IDH1 (Cell Signaling Technology, Danvers, USA #D2H1, 1/1000), LOX (home-made antibody, 1/250), AVEN (Sigma, St-Louis, USA #HPA020863, 1/100) or ACTIN (Millipore, Billerica, USA #MAB 1501 Clone C4, 1/5000).

Techniques: Expressing, Immunofluorescence, Immunohistochemistry, Labeling

Journal: Aging (Albany NY)

Article Title: An expression screen for aged-dependent microRNAs identifies miR-30a as a key regulator of aging features in human epidermis

doi: 10.18632/aging.101326

Figure Lengend Snippet: ( A ) The expression levels of AVEN, IDH1 and LOX proteins were evaluated by western blotting in cultured keratinocytes transduced by the miR-30a lentivirus construction after doxycycline treatment. ( B ) Immuno-fluorescent staining of AVEN and IDH1 and immunohistochemical staining of LOX in reconstructed epidermis overexpressing or not miR-30a after doxycycline treatment. Counterstaining was performed with DAPI and polycarbonate membrane position is indicated by a dotted line. Representative photographs of 3 independent replicates were shown. Scale bar = 25 μm. Right panels: quantification of the IDH1, LOX or AVEN labeled area in the REs Results are mean +/− SD from three independent samples. ****P<0,0001, ***P<0,001 ( C ) The alignment of miR-30a putative binding sites in human AVEN , IDH1 or LOX 3′-UTR region have been schematized to show complementary pairing of miR-30a with AVEN , IDH1 or LOX wild-type (WT) and mutant (Mut) 3′-UTR constructs. Transduced keratinocytes were transfected with WT or Mut reporter constructs. Luciferase intensities were normalized to β-galactosidase level. Results are mean +/− SD from three independent samples. *P<0,05, **P<0,01, ***P<0,001, ****P<0,0001.

Article Snippet: The membrane was incubated overnight at 4°C in TBS-T with primary antibody specific to IDH1 (Cell Signaling Technology, Danvers, USA #D2H1, 1/1000), LOX (home-made antibody, 1/250), AVEN (Sigma, St-Louis, USA #HPA020863, 1/100) or ACTIN (Millipore, Billerica, USA #MAB 1501 Clone C4, 1/5000).

Techniques: Expressing, Western Blot, Cell Culture, Staining, Immunohistochemical staining, Labeling, Binding Assay, Mutagenesis, Construct, Transfection, Luciferase