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: 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: Neuro-Oncology

Article Title: Characterization of single microvesicles in plasma from glioblastoma patients

doi: 10.1093/neuonc/noy187

Figure Lengend Snippet: Marker expression in microvesicles obtained from GBM8. (A) Images of MV-like vesicles labeled for generic EV markers (CD81, TSG101, CD63, Alix, integrin beta 1, CD9, CD40, Arf6, VAMP-3) and glioblastoma markers (EGFR, EGFRvIII, EpCAM, IDH1-R132H) were quantified per single vesicle. Heat map of data is organized by high expression to low expression. Note the heterogeneity profile of each individual MV-like vesicle. (B) Waterfall plot displaying the percent of the total MV-like vesicles that are positive for a given marker. IDH1-R132H was not detected in any of the vesicles.

Article Snippet: 31 CD9(VJ1)-CD405M, TSG101, integrin beta 1 (12G10), epidermal growth factor receptor (EGFR)-AF594, CD31-AF647 CD45, epithelial cell adhesion molecule (EpCAM) (AUA1), and Arf6 (poly) antibodies were purchased from Abcam; CD41, CD42b-AF647, CD40 (5C3), CD40 (3/23), CD9 (MZ3), and isocitrate dehydrogenase 1 (IDH1) (O92H9) antibodies were purchased from Biolegend; CD63 (AHN16.1/16-4-5) antibody was purchased from Ancell; Alix, CD81, and VAMP-3 antibodies were purchased from Santa Cruz; IDH1-R132H, Arf6 (EPR8357), and integrin beta 1 (poly) antibodies were purchased from EMD Millipore; CD235a-AF647 antibody was acquired from BioRad; EGFR variant III (EGFRvIII) and IDH1 (D2H1) antibodies were acquired from Cell Signaling Technologies; CD63 (poly) was acquired from R&D Systems; and EpCAM (G8.8) was acquired from eBioscience.

Techniques: Marker, Expressing, Labeling

Journal: Neuro-Oncology

Article Title: Characterization of single microvesicles in plasma from glioblastoma patients

doi: 10.1093/neuonc/noy187

Figure Lengend Snippet: Summary of marker expression in MV-like fractions obtained from different specimens

Article Snippet: 31 CD9(VJ1)-CD405M, TSG101, integrin beta 1 (12G10), epidermal growth factor receptor (EGFR)-AF594, CD31-AF647 CD45, epithelial cell adhesion molecule (EpCAM) (AUA1), and Arf6 (poly) antibodies were purchased from Abcam; CD41, CD42b-AF647, CD40 (5C3), CD40 (3/23), CD9 (MZ3), and isocitrate dehydrogenase 1 (IDH1) (O92H9) antibodies were purchased from Biolegend; CD63 (AHN16.1/16-4-5) antibody was purchased from Ancell; Alix, CD81, and VAMP-3 antibodies were purchased from Santa Cruz; IDH1-R132H, Arf6 (EPR8357), and integrin beta 1 (poly) antibodies were purchased from EMD Millipore; CD235a-AF647 antibody was acquired from BioRad; EGFR variant III (EGFRvIII) and IDH1 (D2H1) antibodies were acquired from Cell Signaling Technologies; CD63 (poly) was acquired from R&D Systems; and EpCAM (G8.8) was acquired from eBioscience.

Techniques: Marker, Expressing

Journal: Neuro-Oncology

Article Title: Characterization of single microvesicles in plasma from glioblastoma patients

doi: 10.1093/neuonc/noy187

Figure Lengend Snippet: Global marker expression in human GB plasma samples. (A) Total MV-like vesicle numbers in the plasma from each patient were determined via nanoparticle tracking analysis (NTA). (B) The amount of TMV was determined by first accounting for host cell MV (using a cocktail of CD31, CD41, CD42β, CD45, and CD235a) then staining remaining vesicles for tumor markers (EGFR, EGFRvIII, EpCAM, and IDH1-R132H). TMV were present and detectable in all GB patients analyzed. (C) The relative contribution of TMV to the total MV-like vesicle pool was variable among GB patients. (D) The heterogeneity of EGFR, EGFRvIII, and EpCAM expression (IDH1-R132H was not detected in any of the EV observed) among the subpopulation of TMV for each patient was assessed.

Article Snippet: 31 CD9(VJ1)-CD405M, TSG101, integrin beta 1 (12G10), epidermal growth factor receptor (EGFR)-AF594, CD31-AF647 CD45, epithelial cell adhesion molecule (EpCAM) (AUA1), and Arf6 (poly) antibodies were purchased from Abcam; CD41, CD42b-AF647, CD40 (5C3), CD40 (3/23), CD9 (MZ3), and isocitrate dehydrogenase 1 (IDH1) (O92H9) antibodies were purchased from Biolegend; CD63 (AHN16.1/16-4-5) antibody was purchased from Ancell; Alix, CD81, and VAMP-3 antibodies were purchased from Santa Cruz; IDH1-R132H, Arf6 (EPR8357), and integrin beta 1 (poly) antibodies were purchased from EMD Millipore; CD235a-AF647 antibody was acquired from BioRad; EGFR variant III (EGFRvIII) and IDH1 (D2H1) antibodies were acquired from Cell Signaling Technologies; CD63 (poly) was acquired from R&D Systems; and EpCAM (G8.8) was acquired from eBioscience.

Techniques: Marker, Expressing, Staining

Journal: Neuro-Oncology

Article Title: Characterization of single microvesicles in plasma from glioblastoma patients

doi: 10.1093/neuonc/noy187

Figure Lengend Snippet: Patient demographics and clinical characteristics

Article Snippet: 31 CD9(VJ1)-CD405M, TSG101, integrin beta 1 (12G10), epidermal growth factor receptor (EGFR)-AF594, CD31-AF647 CD45, epithelial cell adhesion molecule (EpCAM) (AUA1), and Arf6 (poly) antibodies were purchased from Abcam; CD41, CD42b-AF647, CD40 (5C3), CD40 (3/23), CD9 (MZ3), and isocitrate dehydrogenase 1 (IDH1) (O92H9) antibodies were purchased from Biolegend; CD63 (AHN16.1/16-4-5) antibody was purchased from Ancell; Alix, CD81, and VAMP-3 antibodies were purchased from Santa Cruz; IDH1-R132H, Arf6 (EPR8357), and integrin beta 1 (poly) antibodies were purchased from EMD Millipore; CD235a-AF647 antibody was acquired from BioRad; EGFR variant III (EGFRvIII) and IDH1 (D2H1) antibodies were acquired from Cell Signaling Technologies; CD63 (poly) was acquired from R&D Systems; and EpCAM (G8.8) was acquired from eBioscience.

Techniques:

Journal: Neuro-Oncology

Article Title: Characterization of single microvesicles in plasma from glioblastoma patients

doi: 10.1093/neuonc/noy187

Figure Lengend Snippet: Marker expression in microvesicles obtained from GBM8. (A) Images of MV-like vesicles labeled for generic EV markers (CD81, TSG101, CD63, Alix, integrin beta 1, CD9, CD40, Arf6, VAMP-3) and glioblastoma markers (EGFR, EGFRvIII, EpCAM, IDH1-R132H) were quantified per single vesicle. Heat map of data is organized by high expression to low expression. Note the heterogeneity profile of each individual MV-like vesicle. (B) Waterfall plot displaying the percent of the total MV-like vesicles that are positive for a given marker. IDH1-R132H was not detected in any of the vesicles.

Article Snippet: 31 CD9(VJ1)-CD405M, TSG101, integrin beta 1 (12G10), epidermal growth factor receptor (EGFR)-AF594, CD31-AF647 CD45, epithelial cell adhesion molecule (EpCAM) (AUA1), and Arf6 (poly) antibodies were purchased from Abcam; CD41, CD42b-AF647, CD40 (5C3), CD40 (3/23), CD9 (MZ3), and isocitrate dehydrogenase 1 (IDH1) (O92H9) antibodies were purchased from Biolegend; CD63 (AHN16.1/16-4-5) antibody was purchased from Ancell; Alix, CD81, and VAMP-3 antibodies were purchased from Santa Cruz; IDH1-R132H, Arf6 (EPR8357), and integrin beta 1 (poly) antibodies were purchased from EMD Millipore; CD235a-AF647 antibody was acquired from BioRad; EGFR variant III (EGFRvIII) and IDH1 (D2H1) antibodies were acquired from Cell Signaling Technologies; CD63 (poly) was acquired from R&D Systems; and EpCAM (G8.8) was acquired from eBioscience.

Techniques: Marker, Expressing, Labeling

Journal: Neuro-Oncology

Article Title: Characterization of single microvesicles in plasma from glioblastoma patients

doi: 10.1093/neuonc/noy187

Figure Lengend Snippet: Summary of marker expression in MV-like fractions obtained from different specimens

Article Snippet: 31 CD9(VJ1)-CD405M, TSG101, integrin beta 1 (12G10), epidermal growth factor receptor (EGFR)-AF594, CD31-AF647 CD45, epithelial cell adhesion molecule (EpCAM) (AUA1), and Arf6 (poly) antibodies were purchased from Abcam; CD41, CD42b-AF647, CD40 (5C3), CD40 (3/23), CD9 (MZ3), and isocitrate dehydrogenase 1 (IDH1) (O92H9) antibodies were purchased from Biolegend; CD63 (AHN16.1/16-4-5) antibody was purchased from Ancell; Alix, CD81, and VAMP-3 antibodies were purchased from Santa Cruz; IDH1-R132H, Arf6 (EPR8357), and integrin beta 1 (poly) antibodies were purchased from EMD Millipore; CD235a-AF647 antibody was acquired from BioRad; EGFR variant III (EGFRvIII) and IDH1 (D2H1) antibodies were acquired from Cell Signaling Technologies; CD63 (poly) was acquired from R&D Systems; and EpCAM (G8.8) was acquired from eBioscience.

Techniques: Marker, Expressing

Journal: Neuro-Oncology

Article Title: Characterization of single microvesicles in plasma from glioblastoma patients

doi: 10.1093/neuonc/noy187

Figure Lengend Snippet: Global marker expression in human GB plasma samples. (A) Total MV-like vesicle numbers in the plasma from each patient were determined via nanoparticle tracking analysis (NTA). (B) The amount of TMV was determined by first accounting for host cell MV (using a cocktail of CD31, CD41, CD42β, CD45, and CD235a) then staining remaining vesicles for tumor markers (EGFR, EGFRvIII, EpCAM, and IDH1-R132H). TMV were present and detectable in all GB patients analyzed. (C) The relative contribution of TMV to the total MV-like vesicle pool was variable among GB patients. (D) The heterogeneity of EGFR, EGFRvIII, and EpCAM expression (IDH1-R132H was not detected in any of the EV observed) among the subpopulation of TMV for each patient was assessed.

Article Snippet: 31 CD9(VJ1)-CD405M, TSG101, integrin beta 1 (12G10), epidermal growth factor receptor (EGFR)-AF594, CD31-AF647 CD45, epithelial cell adhesion molecule (EpCAM) (AUA1), and Arf6 (poly) antibodies were purchased from Abcam; CD41, CD42b-AF647, CD40 (5C3), CD40 (3/23), CD9 (MZ3), and isocitrate dehydrogenase 1 (IDH1) (O92H9) antibodies were purchased from Biolegend; CD63 (AHN16.1/16-4-5) antibody was purchased from Ancell; Alix, CD81, and VAMP-3 antibodies were purchased from Santa Cruz; IDH1-R132H, Arf6 (EPR8357), and integrin beta 1 (poly) antibodies were purchased from EMD Millipore; CD235a-AF647 antibody was acquired from BioRad; EGFR variant III (EGFRvIII) and IDH1 (D2H1) antibodies were acquired from Cell Signaling Technologies; CD63 (poly) was acquired from R&D Systems; and EpCAM (G8.8) was acquired from eBioscience.

Techniques: Marker, Expressing, Staining

Journal: Neuro-Oncology

Article Title: Characterization of single microvesicles in plasma from glioblastoma patients

doi: 10.1093/neuonc/noy187

Figure Lengend Snippet: Patient demographics and clinical characteristics

Article Snippet: 31 CD9(VJ1)-CD405M, TSG101, integrin beta 1 (12G10), epidermal growth factor receptor (EGFR)-AF594, CD31-AF647 CD45, epithelial cell adhesion molecule (EpCAM) (AUA1), and Arf6 (poly) antibodies were purchased from Abcam; CD41, CD42b-AF647, CD40 (5C3), CD40 (3/23), CD9 (MZ3), and isocitrate dehydrogenase 1 (IDH1) (O92H9) antibodies were purchased from Biolegend; CD63 (AHN16.1/16-4-5) antibody was purchased from Ancell; Alix, CD81, and VAMP-3 antibodies were purchased from Santa Cruz; IDH1-R132H, Arf6 (EPR8357), and integrin beta 1 (poly) antibodies were purchased from EMD Millipore; CD235a-AF647 antibody was acquired from BioRad; EGFR variant III (EGFRvIII) and IDH1 (D2H1) antibodies were acquired from Cell Signaling Technologies; CD63 (poly) was acquired from R&D Systems; and EpCAM (G8.8) was acquired from eBioscience.

Techniques:

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