Journal: International Journal of Biological Sciences

Article Title: Ugonin inhibits chondrosarcoma metastasis through suppressing cathepsin V via promoting miR-4799-5p expression

doi: 10.7150/ijbs.106827

Figure Lengend Snippet: Ugonin V inhibits chondrosarcoma cell migration and invasion in a concentration-dependent manner. (A) The chemical structure of ugonin V. (B) JJ012 and SW1353 cells were treated with different concentrations of ugonin V (1 μM, 3 μM, and 10 μM) for 24 and 48 h, MTT assay was used to evaluate the cytotoxicity effect. (C&D) Migration and invasion assay determined the migration and invasion ability of JJ012 and SW1353 cells after 24 h incubated in different concentrations of ugonin V (1 μM, 3 μM, 10 μM). * p < 0.05.

Article Snippet: The grade 2 mutant IDH2 SW1353 cell line was purchased from ATCC (Manassas, VA, USA) and the grade 2 mutant IDH1 JJ012 cell line was a gift from Dr. Sean P. Scully (University of Miami School of Medicine in Miami, FL, USA).

Techniques: Migration, Concentration Assay, MTT Assay, Invasion Assay, Incubation

Journal: International Journal of Biological Sciences

Article Title: Ugonin inhibits chondrosarcoma metastasis through suppressing cathepsin V via promoting miR-4799-5p expression

doi: 10.7150/ijbs.106827

Figure Lengend Snippet: Over-expression of CTSV reverses ugonin V effect in chondrosarcoma cell migration and invasion. (A&B) Western blot analysis of CTSV expression in JJ012 and SW1353 after pcDNA3.1(+)/CTSV vector and empty vector were transfected. (C&D) Parental and CTSV-overexpressing JJ012 and SW1353 cell lines were treated with 10 μM ugonin V for 24 h, cell motility was respectively analyzed by migration and invasion assays. * p < 0.05 compared to control group; # p < 0.05 compared to the ugonin V-treated group.

Article Snippet: The grade 2 mutant IDH2 SW1353 cell line was purchased from ATCC (Manassas, VA, USA) and the grade 2 mutant IDH1 JJ012 cell line was a gift from Dr. Sean P. Scully (University of Miami School of Medicine in Miami, FL, USA).

Techniques: Over Expression, Migration, Western Blot, Expressing, Plasmid Preparation, Transfection, Control

Journal: International Journal of Biological Sciences

Article Title: Ugonin inhibits chondrosarcoma metastasis through suppressing cathepsin V via promoting miR-4799-5p expression

doi: 10.7150/ijbs.106827

Figure Lengend Snippet: MiR-4799-5p directly binds to CTSV 3'-UTR to reduce CTSV synthesis and suppress chondrosarcoma cell migration and invasion. (A) The graphic illustrates the binding site of miR-4799-5p on CTSV 3'-UTR predicted by the TargetScan database. (B) Relative luciferase activities of chondrosarcoma cells transfected with a CTSV 3'-UTR luciferase reporter vector, after 24 h of ugonin V treatment. JJ012 and SW1353 cell lines were transfected with a miR-4799-5p inhibitor and negative control, followed by 24 h of ugonin V treatment (10 μM). (C-E) Western blotting and qPCR analysis were used to determine CTSV expression. (F&G) Chondrosarcoma cell motility was analyzed by migration and invasion assays. * p < 0.05 compared to control group; # p < 0.05 compared to the ugonin V-treated group. WT, wildtype; MUT, mutant; 3'-UTR, three prime untranslated regions; NC, negative control.

Article Snippet: The grade 2 mutant IDH2 SW1353 cell line was purchased from ATCC (Manassas, VA, USA) and the grade 2 mutant IDH1 JJ012 cell line was a gift from Dr. Sean P. Scully (University of Miami School of Medicine in Miami, FL, USA).

Techniques: Migration, Binding Assay, Luciferase, Transfection, Plasmid Preparation, Negative Control, Western Blot, Expressing, Control, Mutagenesis

Journal: Cell metabolism

Article Title: A potent and specific CD38 inhibitor ameliorates age-related metabolic dysfunction by reversing tissue NAD + decline

doi: 10.1016/j.cmet.2018.03.016

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Gene Symbol Probe ID Ccl2 Mm00441242_g2 Cd38 Mm01220906_m1 Col1a1 Mm00801666_g1 Cox1 Mm04225243_g1 Fgf21 Mm00840165_g1 G6pc Mm00839363_m1 Gck Mm00439129_m1 Glut1 Mm00441480_m1 Glut4 Mm00441480_m1 Hk1 Mm00439344_m1 Hk2 Mm00443385_m1 Idh2 Mm00612429_m1 Igf1 Mm00439560_m1 Igf1r Mm00802831_m1 Igfbp1 Mm00515154_m1 Il6 Mm00446190_m1 Ldha Mm01612132_g1 Mdh2 Mm00725890_s1 Mmp2 Mm00439498_m1 Nadk Mm00446804_m1 Nampt Mm00451938_m1 Naprt1 Mm01205844_g1 Nd4 Mm04225294_s1 Ndufb5 Mm00452592_m1 Nrf1 Mm01135606_m1 Nrf2 Mm00477784_m1 Pai1 Mm00435860_m1 Parp1 Mm01321084_m1 Parp2 Mm00456462_m1 Pdha1 Mm00468675_m1 Pdk1 Mm00554300_m1 Pkm Mm00834102_gH Pgc1a Mm00447180_m1 Ppara Mm00440939_m1 Sdha Mm01352366_m1 Sirt1 Mm00490758_m1 Sirt3 Mm00452131_m1 Sirt6 Mm01149042_m1 Tgfb1 Mm01178820_m1 Tnfa Mm00443258_m1 Gapdh 4352932E Hprt Mm01545399_m1 Tbp Mm00446971_m1 Open in a separate window TaqMan Gene Expression Assays

Techniques: Control, Recombinant, Transfection, Protease Inhibitor, Enzyme-linked Immunosorbent Assay, Activity Assay, Colorimetric Assay, Reverse Transcription, Luminescence Assay, Derivative Assay, Plasmid Preparation, Modification, Clone Assay, Mutagenesis, Construct, Software

Journal: Cell metabolism

Article Title: A potent and specific CD38 inhibitor ameliorates age-related metabolic dysfunction by reversing tissue NAD + decline

doi: 10.1016/j.cmet.2018.03.016

Figure Lengend Snippet: TaqMan Gene Expression Assays

Article Snippet: Gene Symbol Probe ID Ccl2 Mm00441242_g2 Cd38 Mm01220906_m1 Col1a1 Mm00801666_g1 Cox1 Mm04225243_g1 Fgf21 Mm00840165_g1 G6pc Mm00839363_m1 Gck Mm00439129_m1 Glut1 Mm00441480_m1 Glut4 Mm00441480_m1 Hk1 Mm00439344_m1 Hk2 Mm00443385_m1 Idh2 Mm00612429_m1 Igf1 Mm00439560_m1 Igf1r Mm00802831_m1 Igfbp1 Mm00515154_m1 Il6 Mm00446190_m1 Ldha Mm01612132_g1 Mdh2 Mm00725890_s1 Mmp2 Mm00439498_m1 Nadk Mm00446804_m1 Nampt Mm00451938_m1 Naprt1 Mm01205844_g1 Nd4 Mm04225294_s1 Ndufb5 Mm00452592_m1 Nrf1 Mm01135606_m1 Nrf2 Mm00477784_m1 Pai1 Mm00435860_m1 Parp1 Mm01321084_m1 Parp2 Mm00456462_m1 Pdha1 Mm00468675_m1 Pdk1 Mm00554300_m1 Pkm Mm00834102_gH Pgc1a Mm00447180_m1 Ppara Mm00440939_m1 Sdha Mm01352366_m1 Sirt1 Mm00490758_m1 Sirt3 Mm00452131_m1 Sirt6 Mm01149042_m1 Tgfb1 Mm01178820_m1 Tnfa Mm00443258_m1 Gapdh 4352932E Hprt Mm01545399_m1 Tbp Mm00446971_m1 Open in a separate window TaqMan Gene Expression Assays

Techniques: Gene Expression

Journal: The FASEB Journal

Article Title: IDH2 reprograms mitochondrial dynamics in cancer through a HIF-1α–regulated pseudohypoxic state

doi: 10.1096/fj.201901366r

Figure Lengend Snippet: Figure 1. IDH2 regulation of mitochondrial bioenergetics. A) Prostate adenocarcinoma PC3 cells were transfected with control nontargeting siRNA (siCtrl) or 2 independent IDH2-directed siRNAs (#3 and #5) and analyzed by Western blotting. B) PC3 cells were transfected with siCtrl or IDH1- or IDH2-directed siRNA and analyzed by Western blotting. C, D) PC3 cells transfected as in B were analyzed for OCRs on a Seahorse XFe96 Bioenergetics Flux Analyzer [representative tracings; n = 2 (C)], and basal (left) and maximal (right) respiratory capacities were quantified (D). Means 6 SD (n = 22). *P , 0.01, ***P , 0.0001. E) The conditions are as in B, and transfected PC3 cells were analyzed for ECARs on a Seahorse XFe96 Bioenergetics Flux Analyzer. Representative tracings (n = 2). F) PC3 cells transfected as in B were analyzed for glucose consumption (Glu) or lactate production (Lac). Means 6 SD (n = 6). *P = 0.01, **P = 0.001, ***P , 0.0001. G) The conditions are as in B, and siRNA- transfected PC3 cells were analyzed for the rate of ATP production on a Seahorse XFe96 Bioenergetics Flux Analyzer. Means 6

Article Snippet: Plasmid cDNAs encoding WT IDH2 or IDH2 Arg172Lys (R172K) neomorphic mutant were from Addgene (Watertown, MA, USA).

Techniques: Transfection, Control, Western Blot

Journal: The FASEB Journal

Article Title: IDH2 reprograms mitochondrial dynamics in cancer through a HIF-1α–regulated pseudohypoxic state

doi: 10.1096/fj.201901366r

Figure Lengend Snippet: Figure 2. IDH2 controls mitochondrial dynamics. A) PC3 cells transfected with siCtrl or siIDH2 were analyzed for mitochondrial morphology by confocal laser microscopy. Representative images. Scale bars, 10 mm. Insets, magnification of indicated areas. B) PC3 cells transfected with siCtrl, 3 independent IDH2-directed siRNAs (1, 2, 3), or IDH2-directed pooled siRNA (P) were quantified for mitochondrial volume. Means 6 SD (n = 29–33). *P = 0.01, ***P = 0.0002–0.0003. C) The conditions are as in A, and siRNA-transfected PC3 cells were analyzed for changes in mitochondrial dimension indicative of organelle fusion (.1.3-fold change in mitochondrial volume, positive y scale) or fission (,0.7-fold change in mitochondrial volume, negative y scale), and events were quantified continuously by time-lapse videomicroscopy at the indicated time intervals. Each tracing corresponds to an individual cell. Representative experiment (n = 2). D) The conditions are as in C, and mitochondrial fusion and fission events (60-s interval) were quantified in siRNA-transfected DU145 cells. Means 6 SD (n = 13–14). *P = 0.01, **P = 0.001. E) PC3 cells transfected with siCtrl or siIDH2 were fractionated in total (TE), cytosolic (CE), or mitochondrial (ME) extracts and analyzed by Western blotting. F) PC3 cells transfected as in A were labeled with MitoTracker plus an antibody to Ser616-phosphorylated Drp1 and analyzed for signal colocalization by confocal fluorescence microscopy. Representative images (n = 3). G) The experimental conditions are as in F, and colocalization of Ser616-phosphorylated Drp1 and MitoTracker was quantified with determination of a Pearson’s correlation index (PCI). Means 6 SD (n = 24–31). *P = 0.01, **P = 0.009, ***P , 0.0001. H) PC3 cells transfected with siCtrl or siIDH2 were analyzed for mitochondrial motility in 2D contour plots in the presence or absence of Drp1-directed siRNA (siDrp1). Each tracing corresponds to the movement of an individual mitochondrion. The cutoff velocities for slow-moving or fast-moving (,16 nm/s or .16 nm/s, respectively) mitochondria are indicated. Representative experiment (n = 2). I) The conditions are as in H, and the speed of mitochondrial movements (top; n = 45–50) and total distance traveled by individual mitochondria (bottom; n = 47–50) were quantified. FI, fold increase; siCtrl, control nontargeting siRNA; VDAC, voltage- dependent anion channel. *P = 0.01, **P = 0.001-0.003, ***P , 0.0001.

Article Snippet: Plasmid cDNAs encoding WT IDH2 or IDH2 Arg172Lys (R172K) neomorphic mutant were from Addgene (Watertown, MA, USA).

Techniques: Transfection, Microscopy, Western Blot, Labeling, Control

Journal: The FASEB Journal

Article Title: IDH2 reprograms mitochondrial dynamics in cancer through a HIF-1α–regulated pseudohypoxic state

doi: 10.1096/fj.201901366r

Figure Lengend Snippet: Figure 3. IDH2 regulation of tumor cell movements. A) PC3 cells transfected with siCtrl or siIDH2 were labeled with Talin-Red Fluorescent Protein (RFP) and analyzed for FA complex dynamics by time-lapse videomicroscopy. Representative merged frames at 0 h (magenta) and 2 h (cyan) are shown (n = 2). Arrows, position of new, stable, and decayed FA complexes. B) The conditions are as in A, and the percentage of new, stable, or decayed FA complexes was quantified per each condition (siCtrl, n = 12; siIDH2, n = 7). *P = 0.04. C) PC3 cells stably transduced with shCtrl or 3 independent IDH2-directed shRNAs (D11, D12, E1) were analyzed by Western blotting. P, phosphorylated. D) PC3 cells were transfected with siCtrl or siIDH2 and analyzed for cellular motility in 2D contour plots in the presence or absence of Drp1-directed siRNA (siDrp1). Each tracing corresponds to the movements of an individual cell. The cutoff velocities for slow-moving or fast-moving cells (,0.69 or .0.69 mm/min, respectively) are indicated. E, F) The conditions are as in D, and the speed of cell motility n = 45–66 (E)] and total distance traveled by individual cells [n = 58–66 (F)] was quantified. *P = 0.01, **P = 0.003, ***P , 0.0001. G, H) PC3 cells transfected with siCtrl or siIDH2 were analyzed for directional cell migration in a wound-closure assay (G), and the area covered by cell migration was quantified at the indicated time intervals (H). Representative images (n = 3). BAF, binary area fraction; shCtrl, control nontargeting shRNA; siCtrl, control nontargeting siRNA.

Article Snippet: Plasmid cDNAs encoding WT IDH2 or IDH2 Arg172Lys (R172K) neomorphic mutant were from Addgene (Watertown, MA, USA).

Techniques: Transfection, Labeling, Stable Transfection, Transduction, Western Blot, Migration, Wound Closure Assay, Control, shRNA

Journal: The FASEB Journal

Article Title: IDH2 reprograms mitochondrial dynamics in cancer through a HIF-1α–regulated pseudohypoxic state

doi: 10.1096/fj.201901366r

Figure Lengend Snippet: Figure 4. Requirements IDH2 regulation of tumor cell motility. A) PC3 cells transfected with siCtrl or siIDH2 were analyzed for cell migration (top) or invasion across Matrigel-coated Transwell inserts (bottom). Representative images of DAPI-stained nuclei of migrated or invaded cells are shown. B) PC3 (top, n = 21–25) or DU145 (bottom, n = 22–24) cells were transfected with siCtrl, 3 independent IDH2-directed siRNAs (#1, #2, #3), or IDH2-directed pooled siRNA (P) and analyzed for Matrigel invasion. Means 6 SD. *P = 0.02, ***P , 0.0001. C) PC3 cells transfected as in A were reconstituted with IDH2 cDNA and analyzed for cell migration (top, n = 21–22) or Matrigel invasion (bottom, n = 21–22). Means 6 SD. ***P , 0.0001. D, E) PC3 cells transfected with siCtrl or siIDH2 were further transfected with Drp1-directed siRNA (siDrp1) and analyzed by Western blotting (D) or Matrigel invasion [n = 37–45 (E)]. Means 6 SD. ***P , 0.0001. F, G) PC3 cells transfected with siCtrl or siIDH2 were incubated with small molecule Akt inhibitor, MK2206, and analyzed by Western blotting (F) or Matrigel invasion (G). siCtrl, control nontargeting siRNA; ns, not significant; p, phosphorylated. Means 6 SD (n = 22–24). ***P , 0.0001.

Article Snippet: Plasmid cDNAs encoding WT IDH2 or IDH2 Arg172Lys (R172K) neomorphic mutant were from Addgene (Watertown, MA, USA).

Techniques: Transfection, Migration, Staining, Western Blot, Incubation, Control

Journal: The FASEB Journal

Article Title: IDH2 reprograms mitochondrial dynamics in cancer through a HIF-1α–regulated pseudohypoxic state

doi: 10.1096/fj.201901366r

Figure Lengend Snippet: Figure 5. ROS regulation of IDH2-directed tumor cell motil- ity. A) PC3 cells transfected with siCtrl or siIDH2 were reconsti- tuted with Prx3 or loss-of-func- tion Cys108Ser (C108S) Prx3 mutant (Prx3-Mut) and quanti- fied for speed of mitochondrial movements (top, n = 85–92) or distance traveled by individual mitochondria (bottom, n = 85–92). ***P , 0.0001. B) The conditions are as in A, and reconstituted PC3 cells were analyzed for cell motility in 2D contour plots. Each tracing corresponds to the movement of an individual cell. The cutoff velocities for slow-moving or fast-moving (,0.39 mm/min or .0.39 mm/min, respectively) cells are shown. Representative experiment (n = 3). C, D) PC3 cells transfected with siCtrl or siIDH2 and reconstituted as in A were analyzed for cell migra- tion [top n = 10–14 (C)] or Matrigel invasion [bottom n = 10–12 (C)] or Western blotting (D). Means 6 SD. ***P , 0.0001. E) PC3 cells transfected with siCtrl or siIDH2 were in- cubated with the ROS scaven- ger, MnTBAP, and analyzed by Western blotting. Ns, not signif- icant; p, phosphorylated.

Article Snippet: Plasmid cDNAs encoding WT IDH2 or IDH2 Arg172Lys (R172K) neomorphic mutant were from Addgene (Watertown, MA, USA).

Techniques: Transfection, Mutagenesis, Western Blot

Journal: The FASEB Journal

Article Title: IDH2 reprograms mitochondrial dynamics in cancer through a HIF-1α–regulated pseudohypoxic state

doi: 10.1096/fj.201901366r

Figure Lengend Snippet: Figure 6. HIF-1a regulation by IDH2. A) PC3 (left) or DU145 (right) cells transfected with siCtrl, 3 independent IDH2-directed siRNAs (1, 2, 3), or pooled IDH2-directed siRNA (P) were analyzed by Western blotting. B) PC3 cells stably transduced with 2 independent control shRNAs (shCtrl #1 and #2) or 3 IDH2-directed shRNAs (D11, D12, and E1) were analyzed by Western blotting. C) PC3 cells transfected with siCtrl, siIDH1, or siIDH2 were analyzed by Western blotting. D) PC3 cells transfected with siCtrl or siIDH2 were reconstituted with vector or IDH2 cDNA and analyzed by Western blotting. E) PC3 cells transfected as in C were analyzed at the indicated time intervals by Western blotting. Bar graph (bottom), densitometric quantification of HIF-1a protein bands. F) PC3 (top) or DU145 (bottom) cells transfected with siCtrl or siIDH2 were incubated with the ROS scavenger, MnTBAP, and analyzed by Western blotting. G) PC3 cells transfected with siCtrl or siIDH2 were reconstituted with Prx3 or loss-of- function C108S Prx3 mutant (Prx3-Mut) and analyzed by Western blotting. H) PC3 cells transfected with siCtrl, siIDH2, or siHIF- 1a were analyzed for cell motility in 2D contour plots. Each line corresponds to the movements of an individual cell. The cutoff velocities for slow-moving or fast-moving (,0.45 mm/min or .0.45 mm/min) cells are indicated. Representative experiment (n = 2). I) The conditions are as in H, and the speed of cell movements (top, n = 58–64) and total distance traveled by individual cells (bottom, n = 58–64) was quantified per each condition. *P = 0.01, ***P , 0.0001. J) The conditions are as in H, and siRNA- transfected PC3 cells were analyzed for cell migration (top, n = 13–16) or Matrigel invasion (bottom, n = 23–31). Ns, not significant; shCtrl, control nontargeting shRNA; siCtrl, control nontargeting siRNA. Means 6 SD. ***P = 0.0002 to P , 0.0001.

Article Snippet: Plasmid cDNAs encoding WT IDH2 or IDH2 Arg172Lys (R172K) neomorphic mutant were from Addgene (Watertown, MA, USA).

Techniques: Transfection, Western Blot, Stable Transfection, Transduction, Control, Plasmid Preparation, Incubation, Mutagenesis, Migration, shRNA

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: TaqMan quantitative RT-PCR (qRT-PCR; Life Technologies) was performed for IDH2 (Hs00158033_m1), Twist1 (Hs01675818_s1), SNAI1 (Hs00195591_m1), SNAI2 (Hs00161904_m1), FN1 (Hs01549976_m1), and using GAPDH (Hs99999905_m1) for expression assay normalization, as previously described ( 5 , 20 ).

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: TaqMan quantitative RT-PCR (qRT-PCR; Life Technologies) was performed for IDH2 (Hs00158033_m1), Twist1 (Hs01675818_s1), SNAI1 (Hs00195591_m1), SNAI2 (Hs00161904_m1), FN1 (Hs01549976_m1), and using GAPDH (Hs99999905_m1) for expression assay normalization, as previously described ( 5 , 20 ).

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

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: Journal of Hematology & Oncology

Article Title: Reductive TCA cycle catalyzed by wild-type IDH2 promotes acute myeloid leukemia and is a metabolic vulnerability for potential targeted therapy

doi: 10.1186/s13045-022-01245-z

Figure Lengend Snippet: Over-expression of wild-type IDH2 in AML and its effect on leukemia cell proliferation. a Comparison of IDH2 mRNA levels in AML ( n = 80) and normal cells (monocytes and neutrophils, n = 6), using the leukemia dataset (Stegmaier Leukemia datasets) available in the Oncomine database. b IDH2 mRNA levels in primary AML samples ( n = 204) in comparison with normal cells (hematopoietic stem cells, n = 6; metamyelocytes, n = 3; band cells, n = 3; polymorphonuclear cells, n = 3; monocytes, n = 4) available in the BloodSpot datasets. c Western blotting of IDH2 in normal human peripheral blood mononuclear cells (PBMC #1–#3), AML cell lines and PBMCs from AML patients with wt-IDH2 (AML#1–#8), β-actin was used as a loading control. d Relative mRNA level of IDH2 in U937 and ML-1 cells transfected with control shRNA (shRNA-Ctrl) or IDH2 shRNA (shIDH2#1, shIDH2#2) was measured by RT-qPCR. e Western blotting analysis of IDH2 protein in U937 and ML-1 cells transfected with shRNA-Ctrl or IDH2 shRNA. f , g Measurement of cell proliferation in U937 and ML-1 cells expressing shRNA-Ctrl or IDH2 shRNA. h Relative IDH2 mRNA level in HL-60 cells transfected with control vector (Vector) or IDH2 over-expression vector (IDH2 OE ). Expression of mRNA was measured by RT-qPCR. i Western blotting analysis of IDH2 protein levels in HL-60 cells transfected with control vector or IDH2 over-expression vector. j Comparison of cell proliferation in HL-60 cells transfected with control vector or IDH2 over-expression vector. k , l Colony formation assay in U937 and ML-1 cells expressing shRNA-Ctrl or IDH2 shRNA. Colonies were counted under inverted microscope. m Comparison of percentage of Annexin V/ PI negative cells in U937 and ML-1 transfected with shRNA-Ctrl or IDH2 shRNA for 48 h. The original flow cytometry analysis data are presented as Additional file : Fig. S1h. n = 3, mean ± SD ; * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: In brief, 20,000 cells were seeded in a 96-well plate and treated with the indicated doses of IDH2 inhibitor, AGI-6780 (Selleck, Houston, TX77014, USA) for 72 h and stained for 4 h. The optical density at 490 nm was determined using a Multiskan plate reader (Thermo Fisher Scientific).

Techniques: Over Expression, Comparison, Western Blot, Control, Transfection, shRNA, Quantitative RT-PCR, Expressing, Plasmid Preparation, Colony Assay, Inverted Microscopy, Flow Cytometry

Journal: Journal of Hematology & Oncology

Article Title: Reductive TCA cycle catalyzed by wild-type IDH2 promotes acute myeloid leukemia and is a metabolic vulnerability for potential targeted therapy

doi: 10.1186/s13045-022-01245-z

Figure Lengend Snippet: Impact of IDH2 knockdown on AML growth in vivo. a Outline of in vivo study design using AML cell models (U937 and ML-1) with shRNA-Ctrl and shIDH2#1 or shIDH2#2. b – d Tumor growth in athymic nude mice inoculated with U937 cells harboring shRNA-Ctrl, shIDH2#1 or shIDH2#2 ( n = 5 per group, mean ± SEM ). Tumor sizes were measured every two days. At the end of the experiment, tumors were isolated, photographed and weighted. e – g Tumor growth in athymic nude mice inoculated with ML-1 cells harboring shRNA-Ctrl, shIDH2#1 or shIDH2#2 ( n = 7 per group, mean ± SD ). Tumor sizes were measured every two days. Tumors were photographed and weighted at the end of the experiment. The star * in f indicates no tumor formation in this mouse. h Representative Western blotting of IDH2 protein expression in tumor tissues isolated from mice inoculated with AML cells harboring shRNA-Ctrl or IDH2 shRNA as indicated. i Left panel: representative images of Ki-67 IHC staining of tumor tissues from U937 xenografts with shRNA-Ctrl or IDH2 shRNA as indicated. The scale bars represent 50 μm; Right panel: apoptotic cells in tumor tissues of U937 xenografts with shRNA-Ctrl or IDH2 shRNA detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The scale bars represent 100 μm. *** p < 0.001

Article Snippet: In brief, 20,000 cells were seeded in a 96-well plate and treated with the indicated doses of IDH2 inhibitor, AGI-6780 (Selleck, Houston, TX77014, USA) for 72 h and stained for 4 h. The optical density at 490 nm was determined using a Multiskan plate reader (Thermo Fisher Scientific).

Techniques: Knockdown, In Vivo, shRNA, Isolation, Western Blot, Expressing, Immunohistochemistry, TUNEL Assay

Journal: Journal of Hematology & Oncology

Article Title: Reductive TCA cycle catalyzed by wild-type IDH2 promotes acute myeloid leukemia and is a metabolic vulnerability for potential targeted therapy

doi: 10.1186/s13045-022-01245-z

Figure Lengend Snippet: Suppression of IDH2 blocked the conversion of α-KG to isocitrate in reductive TCA cycle. a , b Quantitation of α-KG in U937 or ML-1 cells harboring shRNA-Ctrl, shIDH2#1 or shIDH2#2. c , d Levels of isocitrate in U937 or ML-1 cells harboring shRNA-Ctrl, shIDH2#1 or shIDH2#2. e , f Levels of citrate in U937 or ML-1 cells harboring shRNA-Ctrl, shIDH2#1 or shIDH2#2. g Schematic illustration of changes in α-KG, isocitrate, and citrate in AML cells after suppression of active reductive TCA flow using IDH2 shRNA. h Quantitation of α-KG in tumor tissues from mice bearing U937 xenografts harboring shRNA-Ctrl, shIDH2#1 or shIDH2#2. i , j Comparison of α-KG levels in U937 and ML-1 cells cultured with or without glutamine for 24 h. Gln (+): glutamine 2 mM, Gln (−): glutamine free. n = 3, mean ± SD , * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: In brief, 20,000 cells were seeded in a 96-well plate and treated with the indicated doses of IDH2 inhibitor, AGI-6780 (Selleck, Houston, TX77014, USA) for 72 h and stained for 4 h. The optical density at 490 nm was determined using a Multiskan plate reader (Thermo Fisher Scientific).

Techniques: Quantitation Assay, shRNA, Comparison, Cell Culture

Journal: Journal of Hematology & Oncology

Article Title: Reductive TCA cycle catalyzed by wild-type IDH2 promotes acute myeloid leukemia and is a metabolic vulnerability for potential targeted therapy

doi: 10.1186/s13045-022-01245-z

Figure Lengend Snippet: Glutamine metabolic flux analysis in AML cells with or without IDH2 knockdown. a Schematic of [U- 13 C 5 ] glutamine metabolism tracing experiment. Open circles depict 12 C atoms and filled circles depict 13 C atoms. Metabolite abbreviations: Gln: glutamine, Glu: glutamate, α-KG: alpha-ketoglutarate, Isocit: isocitrate, Cit: citrate, AcCoA: acetyl-coenzyme A, FAs: fatty acid synthesis, Oac: oxaloacetate, Mal: malate, Fum: fumarate, Suc: succinate, Pyr: pyruvate, Asp: aspartate. The red arrows indicate the direction of changes in metabolites due to suppression of reductive TCA flow, while the blue arrows indicate the direction of changes in metabolites of the oxidative TCA segment. b – f Mass isotopomer distribution of the TCA cycle metabolites in U937 cells harboring shRNA-Ctrl or IDH2 shRNA cultured in medium containing [U- 13 C 5 ] glutamine for 24 h. g Mass isotopomer distribution of palmitate in U937 cells with shRNA-Ctrl or IDH2 shRNA after cultured in medium containing [U- 13 C 5 ] glutamine for 24 h. h Percentage of newly synthesized palmitate, oleate and stearate in U937 cells with shRNA-Ctrl or IDH2 shRNA after cultured in medium containing [U- 13 C 5 ] glutamine for 24 h. n = 3, mean ± SD , * p < 0.05, *** p < 0.001

Article Snippet: In brief, 20,000 cells were seeded in a 96-well plate and treated with the indicated doses of IDH2 inhibitor, AGI-6780 (Selleck, Houston, TX77014, USA) for 72 h and stained for 4 h. The optical density at 490 nm was determined using a Multiskan plate reader (Thermo Fisher Scientific).

Techniques: Knockdown, shRNA, Cell Culture, Synthesized

Journal: Journal of Hematology & Oncology

Article Title: Reductive TCA cycle catalyzed by wild-type IDH2 promotes acute myeloid leukemia and is a metabolic vulnerability for potential targeted therapy

doi: 10.1186/s13045-022-01245-z

Figure Lengend Snippet: Cytotoxic effect of α-KG in AML cells with wild-type IDH2. a Induction of apoptosis by cell-permeable DM-αKG in AML cell lines (U937 and ML-1) and primary AML cells isolated from patients with wt-IDH2 (AML#1 and AML#2). Cells were treated with the indicated concentrations of DM-αKG for 48 h, and apoptosis was measured by flow cytometry analysis of annexin-V positivity. The number inside each panel shows the percentage of dead cells. b , c Quantitation of the concentration-dependent apoptosis induced by DM-αKG in U937 and ML-1 cells ( n = 3, mean ± SD ). d Apoptosis of human primary AML cells harboring wild-type ( n = 7) or mutant IDH2 (IDH2-R140Q, n = 2) treated with various concentrations (2, 4, 6, 8 and 10 mM) of DM-αKG for 48 h. Apoptosis was measured by flow cytometry analysis of annexin-V positivity

Article Snippet: In brief, 20,000 cells were seeded in a 96-well plate and treated with the indicated doses of IDH2 inhibitor, AGI-6780 (Selleck, Houston, TX77014, USA) for 72 h and stained for 4 h. The optical density at 490 nm was determined using a Multiskan plate reader (Thermo Fisher Scientific).

Techniques: Isolation, Flow Cytometry, Quantitation Assay, Concentration Assay, Mutagenesis

Journal: Journal of Hematology & Oncology

Article Title: Reductive TCA cycle catalyzed by wild-type IDH2 promotes acute myeloid leukemia and is a metabolic vulnerability for potential targeted therapy

doi: 10.1186/s13045-022-01245-z

Figure Lengend Snippet: Regulation of c-Myc expression by wild-type IDH2 in AML cells. a Effect of IDH2 knockdown on the expression of IDH2, C-MYC and its target gene Bcl-2 in U937 and ML-1 cells. Protein expression was analyzed by Western blotting. b Relative protein levels of C-MYC in U937 and ML-1 cells transfected with shRNA-Ctrl or shIDH2 vectors (#1 and #2). Data are representative of three separate experiments. c Relative mRNA level of C-MYC in U937 cells harboring shRNA-Ctrl or shIDH2 vectors. RNA expression was measured by RT-qPCR. d Relative mRNA level of C-MYC in ML-1 cells harboring shRNA-Ctrl or shIDH2 vectors. RNA expression was measured by RT-qPCR. e Expression of IDH2 protein and C-MYC protein in HL-60 cells transfected with control vector (Vector) or wt-IDH2 expression vector (IDH2 OE ), protein expression was detected by Western blotting. f Relative mRNA levels of C-MYC in HL-60 cells harboring control vector or IDH2 OE vector, measured by RT-qPCR. g Representative images of C-MYC immunohistochemistry (IHC) staining of tumor tissues from U937 xenografts harboring shRNA-Ctrl or shIDH2 vectors (#1 or #2). The scale bars represent 100 μm. n = 3, mean ± SD , * p < 0.05, *** p < 0.001

Article Snippet: In brief, 20,000 cells were seeded in a 96-well plate and treated with the indicated doses of IDH2 inhibitor, AGI-6780 (Selleck, Houston, TX77014, USA) for 72 h and stained for 4 h. The optical density at 490 nm was determined using a Multiskan plate reader (Thermo Fisher Scientific).

Techniques: Expressing, Knockdown, Western Blot, Transfection, shRNA, RNA Expression, Quantitative RT-PCR, Control, Plasmid Preparation, Immunohistochemistry

Journal: Journal of Hematology & Oncology

Article Title: Reductive TCA cycle catalyzed by wild-type IDH2 promotes acute myeloid leukemia and is a metabolic vulnerability for potential targeted therapy

doi: 10.1186/s13045-022-01245-z

Figure Lengend Snippet: Effect of α-KG on c-Myc expression in AML cell lines and primary leukemia cells. a Western blotting analysis of IDH2 and C-MYC protein expression in U937 and ML-1 cells treated with the indicated concentrations of DM-αKG for 6, 12 and 24 h. b Relative protein levels of C-MYC in AML cell lines (data from three separate experiments) and primary leukemia cells from AML patients with wild-type IDH2 ( n = 4) treated in vitro with the indicated concentrations of DM-αKG for 12 h. * p < 0.05, ** p < 0.01, *** p < 0.001. c Relative mRNA level of C-MYC in U937 and ML-1 cells treated with the indicated concentrations of DM-αKG for 6 h. RNA expression was measured by RT-qPCR. *** p < 0.001. d Effect of DM-αKG (4 mM) on c-Myc mRNA stability. Cells were pre-treated with DM-αKG for 4.5 h followed by incubation with 5 μg/mL actinomycin D for the indicated time periods (0, 15, 30, 45, 60 and 90 min). The mRNA degradation rate was estimated according to a published method . e C-MYC protein expression in U937 and ML-1 cells cultured with or without glutamine (Gln) for 24–48 h as indicated. f Western blotting of IDH2 and C-MYC protein expression in primary leukemia cells isolated from 6 AML patients with wild-type IDH2 treated ex-vivo with the indicated concentrations of DM-αKG for 6, 12 and 24 h. g Western blotting analysis of IDH2 and C-MYC protein expression in human primary AML cells from two patients with mutant IDH2 treated ex-vivo with indicated concentrations of DM-αKG for 6, 12 and 24 h

Article Snippet: In brief, 20,000 cells were seeded in a 96-well plate and treated with the indicated doses of IDH2 inhibitor, AGI-6780 (Selleck, Houston, TX77014, USA) for 72 h and stained for 4 h. The optical density at 490 nm was determined using a Multiskan plate reader (Thermo Fisher Scientific).

Techniques: Expressing, Western Blot, In Vitro, RNA Expression, Quantitative RT-PCR, Incubation, Cell Culture, Isolation, Ex Vivo, Mutagenesis

Journal: Journal of Hematology & Oncology

Article Title: Reductive TCA cycle catalyzed by wild-type IDH2 promotes acute myeloid leukemia and is a metabolic vulnerability for potential targeted therapy

doi: 10.1186/s13045-022-01245-z

Figure Lengend Snippet: Inhibition of IDH2 by AGI-6780 suppressed AML survival in vitro and in vivo. a , b Effect of AGI-6780 on cell proliferation in U937 and ML-1 cells. Cells were treated with AGI-6780 (10 µM) or solvent (DMSO), and cell numbers were counted at the indicated time points. Data are mean ± SD of three experiments. c Levels of protein expression of IDH2 and C-MYC in U937 and ML-1 cells treated with AGI-6780 (10 µM) or solvent (DMSO) for 24 h. d The indicated cell line were treated with AGI-6780 for 72 h, and cell viability was measured using MTS assay. e Primary leukemia cells from AML patients with wt-IDH2 ( n = 4) and normal human bone marrow cells (HBMC) were treated ex vivo with the indicated concentrations of AGI-6780 for 72 h, and cell viability was measured using MTS assay. f Comparison of percentage of Annexin V/PI-negative cells in primary AML cells and primary normal bone marrow cells treated with 10–20 µM AGI-6780 for 48 h. The original flow cytometry analysis data are presented as Additional file : Fig. S8c, d. g Protein levels of IDH2 and C-MYC in primary AML cells treated with AGI-6780 (20 µM) or with solvent (DMSO) for 24 h. h Growth curves of AML xenografts in athymic nude mice inoculated with ML-1 cells harboring wt-IDH2 ( n = 6 per group, mean ± SEM ). Mice were treated with daily i.p. injection of AGI-6780 or solvent as indicated. i Photographs of gross tumors isolated from mice at the end of the experiment. j Western blot analysis of C-MYC expression in tumor tissues of ML-1 xenografts isolated from mice treated with AGI-6780 or solvent as indicated. k Tumor weights of each group at the end of the experiment ( mean ± SEM ). l Relative C-MYC protein levels in tumor tissues of mice treated with solvent ( n = 6) or AGI-6780 ( n = 6). m Mouse body weights ( mean ± SD ). Two-tailed unpaired student’s t test for a , b , f , h , k and l , * p < 0.05, *** p < 0.001

Article Snippet: In brief, 20,000 cells were seeded in a 96-well plate and treated with the indicated doses of IDH2 inhibitor, AGI-6780 (Selleck, Houston, TX77014, USA) for 72 h and stained for 4 h. The optical density at 490 nm was determined using a Multiskan plate reader (Thermo Fisher Scientific).

Techniques: Inhibition, In Vitro, In Vivo, Solvent, Expressing, MTS Assay, Ex Vivo, Comparison, Flow Cytometry, Injection, Isolation, Western Blot, Two Tailed Test

Journal: Journal of Hematology & Oncology

Article Title: Reductive TCA cycle catalyzed by wild-type IDH2 promotes acute myeloid leukemia and is a metabolic vulnerability for potential targeted therapy

doi: 10.1186/s13045-022-01245-z

Figure Lengend Snippet: Schematic model depicting the key functions of wild-type IDH2 in AML cells and the impact of IDH2 suppression on α-KG metabolism, fatty acid synthesis, and c-Myc expression (see text for detail description)

Article Snippet: In brief, 20,000 cells were seeded in a 96-well plate and treated with the indicated doses of IDH2 inhibitor, AGI-6780 (Selleck, Houston, TX77014, USA) for 72 h and stained for 4 h. The optical density at 490 nm was determined using a Multiskan plate reader (Thermo Fisher Scientific).

Techniques: Expressing

Journal: Cell metabolism

Article Title: A potent and specific CD38 inhibitor ameliorates age-related metabolic dysfunction by reversing tissue NAD + decline

doi: 10.1016/j.cmet.2018.03.016

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mitochondrial copy number was calculated as the ratio of Cox1 or Nd4 to Gapdh . table ft1 table-wrap mode="anchored" t5 caption a7 Gene Symbol Probe ID Ccl2 Mm00441242_g2 Cd38 Mm01220906_m1 Col1a1 Mm00801666_g1 Cox1 Mm04225243_g1 Fgf21 Mm00840165_g1 G6pc Mm00839363_m1 Gck Mm00439129_m1 Glut1 Mm00441480_m1 Glut4 Mm00441480_m1 Hk1 Mm00439344_m1 Hk2 Mm00443385_m1 Idh2 Mm00612429_m1 Igf1 Mm00439560_m1 Igf1r Mm00802831_m1 Igfbp1 Mm00515154_m1 Il6 Mm00446190_m1 Ldha Mm01612132_g1 Mdh2 Mm00725890_s1 Mmp2 Mm00439498_m1 Nadk Mm00446804_m1 Nampt Mm00451938_m1 Naprt1 Mm01205844_g1 Nd4 Mm04225294_s1 Ndufb5 Mm00452592_m1 Nrf1 Mm01135606_m1 Nrf2 Mm00477784_m1 Pai1 Mm00435860_m1 Parp1 Mm01321084_m1 Parp2 Mm00456462_m1 Pdha1 Mm00468675_m1 Pdk1 Mm00554300_m1 Pkm Mm00834102_gH Pgc1a Mm00447180_m1 Ppara Mm00440939_m1 Sdha Mm01352366_m1 Sirt1 Mm00490758_m1 Sirt3 Mm00452131_m1 Sirt6 Mm01149042_m1 Tgfb1 Mm01178820_m1 Tnfa Mm00443258_m1 Gapdh 4352932E Hprt Mm01545399_m1 Tbp Mm00446971_m1 Open in a separate window TaqMan Gene Expression Assays Liver and Skeletal Muscle Immunohistochemistry Liver OCT-embedded liver tissue was cryosectioned at 10 μm thickness, fixed in MeOH:acetone (1:1) for 5 min at −20°C.

Techniques: Control, Recombinant, Transfection, Protease Inhibitor, Enzyme-linked Immunosorbent Assay, Activity Assay, Colorimetric Assay, Reverse Transcription, Luminescence Assay, Derivative Assay, Plasmid Preparation, Modification, Clone Assay, Mutagenesis, Construct, Software

Journal: Cell metabolism

Article Title: A potent and specific CD38 inhibitor ameliorates age-related metabolic dysfunction by reversing tissue NAD + decline

doi: 10.1016/j.cmet.2018.03.016

Figure Lengend Snippet: TaqMan Gene Expression Assays

Article Snippet: Mitochondrial copy number was calculated as the ratio of Cox1 or Nd4 to Gapdh . table ft1 table-wrap mode="anchored" t5 caption a7 Gene Symbol Probe ID Ccl2 Mm00441242_g2 Cd38 Mm01220906_m1 Col1a1 Mm00801666_g1 Cox1 Mm04225243_g1 Fgf21 Mm00840165_g1 G6pc Mm00839363_m1 Gck Mm00439129_m1 Glut1 Mm00441480_m1 Glut4 Mm00441480_m1 Hk1 Mm00439344_m1 Hk2 Mm00443385_m1 Idh2 Mm00612429_m1 Igf1 Mm00439560_m1 Igf1r Mm00802831_m1 Igfbp1 Mm00515154_m1 Il6 Mm00446190_m1 Ldha Mm01612132_g1 Mdh2 Mm00725890_s1 Mmp2 Mm00439498_m1 Nadk Mm00446804_m1 Nampt Mm00451938_m1 Naprt1 Mm01205844_g1 Nd4 Mm04225294_s1 Ndufb5 Mm00452592_m1 Nrf1 Mm01135606_m1 Nrf2 Mm00477784_m1 Pai1 Mm00435860_m1 Parp1 Mm01321084_m1 Parp2 Mm00456462_m1 Pdha1 Mm00468675_m1 Pdk1 Mm00554300_m1 Pkm Mm00834102_gH Pgc1a Mm00447180_m1 Ppara Mm00440939_m1 Sdha Mm01352366_m1 Sirt1 Mm00490758_m1 Sirt3 Mm00452131_m1 Sirt6 Mm01149042_m1 Tgfb1 Mm01178820_m1 Tnfa Mm00443258_m1 Gapdh 4352932E Hprt Mm01545399_m1 Tbp Mm00446971_m1 Open in a separate window TaqMan Gene Expression Assays Liver and Skeletal Muscle Immunohistochemistry Liver OCT-embedded liver tissue was cryosectioned at 10 μm thickness, fixed in MeOH:acetone (1:1) for 5 min at −20°C.

Techniques: Gene Expression

Journal: eLife

Article Title: Replication Study: 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.26030

Figure Lengend Snippet: IDH oxidative and reductive activity assays were performed on lysates of HEK293T cells transfected with wild-type or R172K mutant IDH2, or empty vector. ( A ) Lysates were assessed for generation of NADPH from NADP+ in the presence of 0.4 mM isocitrate over the indicated time course. Mean change in OD 340 from the beginning of each assay (time = 0) is reported for each biological repeat performed [n = 7] and error bars represent s.e.m. ( B ) Linear regression slopes were determined for each biological repeat of the isocitrate-dependent NADPH production assay. Box and whisker plot with median represented as the line through the box and whiskers representing values within 1.5 IQR of the first and third quartile. Means as black dot and bold error bars represent 95% CI. Linear regression slope determined from the data estimated from the representative experiment reported in Figure 2A of is displayed as a single point (red circle) for comparison. Statistical analysis was performed on data generated during this replication attempt. Wild-type IDH2 compared to vector control: Wilcoxon-Mann-Whitney test; Z = 3.13, uncorrected p =0.00058, Bonferroni corrected p =0.0023. R172K mutant IDH2 compared to vector control: Wilcoxon-Mann-Whitney test; Z = 1.60, uncorrected p =0.13, Bonferroni corrected p =0.51. ( C ) Lysates were assessed for consumption of NADPH in the presence of 1 mM alpha-ketoglutarate over the indicated time course. Mean change in OD 340 from the beginning of each assay (time = 0) is reported for each biological repeat performed [n = 7] and error bars represent s.e.m. ( D ) Linear regression slopes were determined for each biological repeat of the alpha-ketoglutarate-dependent NADPH consumption assay. Box and whisker plot with median represented as the line through the box and whiskers representing values within 1.5 IQR of the first and third quartile. Means as black dot and bold error bars represent 95% CI. Linear regression slope determined from the data estimated from the representative experiment reported in Figure 2B of is displayed as a single point (red circle) for comparison. Statistical analysis was performed on data generated during this replication attempt. Wild-type IDH2 compared to vector control: Welch’s t -test; t (9.42) = 0.29, uncorrected p =0.78, Bonferroni corrected p >0.99. R172K mutant IDH2 compared to vector control: Welch’s t -test; t (6.95) = 5.97, uncorrected p =0.00058, Bonferroni corrected p =0.0023. ( E ) Representative Western blots probed with an anti-IDH1 antibody, an anti-IDH2 antibody, and an anti-alpha-Tubulin antibody. Additional details for this experiment can be found at https://osf.io/6ve4d/ . DOI: http://dx.doi.org/10.7554/eLife.26030.002

Article Snippet: To generate pcDNA3.1-IDH2 WT (deposited in Addgene, plasmid# 87926) and pcDNA3.1-IDH2 R172K (deposited in Addgene, plasmid# 87927), IDH2 WT and IDH2 R172K were amplified by PCR from pCMV6-Entry vectors (Origene, Rockville, MD, cat# RC201152 and cat# RC400103) and cloned into the BamH1 and EcoR1 sites of the pcDNA3.1 expression vector (Invitrogen, Carlsbad, CA, cat# V790-20).

Techniques: Activity Assay, Transfection, Mutagenesis, Plasmid Preparation, Whisker Assay, Comparison, Generated, Control, MANN-WHITNEY, Western Blot

Journal: eLife

Article Title: Replication Study: 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.26030

Figure Lengend Snippet: HEK293T cells transfected with wild-type or R172K mutant IDH2, or empty vector, were analyzed for intracellular metabolites. ( A ) Cells harvested 48 hr after transfection had organic acids extracted, purified, and derivatized with MTBSTFA before analysis by GC-MS. Quantitation of 2HG signal intensity relative to glutamate was determined using the TIC for each biological repeat [n = 10]. Box and whisker plot with median represented as the line through the box and whiskers representing values within 1.5 IQR of the first and third quartile. Means as black dot and bold error bars represent 95% CI. Data estimated from the representative experiment reported in Figure 3D of is displayed as a single point (red circle) for comparison. Statistical analysis was performed on log 10 transformed data generated during this replication attempt. R172K mutant IDH2 values were compared to the largest value observed in cells expressing vector control or wild-type IDH2 (0.005). One-sample t -test; t (9) = 21.9, uncorrected p =4.14×10 −9 , Bonferroni corrected p =8.28×10 −9 . ( B ) Representative TIC from samples harvested at 48 hr after transfection for vector control (top panel), wild-type IDH2 (middle panel), and R172K mutant IDH2 (bottom panel). The derivatized organic acids eluting between 31.4 and 34.4 min are shown, including aspartate (31.7 min), 2HG (33.1 min), and glutamate (34.3 min) based on spectra of derivatized commercial standards. ( C ) Cells harvested 24 hr after transfection were processed and analyzed similar to the 48 hr samples with [n = 10] biological repeats. Box and whisker plot with median represented as the line through the box and whiskers representing values within 1.5 IQR of the first and third quartile. Means as black dot and bold error bars represent 95% CI. R172K mutant IDH2 values were compared to the largest value observed in cells expressing vector control or wild-type IDH2 (0.001). One-sample Wilcoxon signed-rank test on log 10 transformed data; V = 55, uncorrected p =0.0020, Bonferroni corrected p =0.0039. ( D ) Representative TIC from samples harvested at 24 hr after transfection for vector control (top panel), wild-type IDH2 (middle panel), and R172K mutant IDH2 (bottom panel). Additional details for this experiment can be found at https://osf.io/9ge2a/ . DOI: http://dx.doi.org/10.7554/eLife.26030.003

Article Snippet: To generate pcDNA3.1-IDH2 WT (deposited in Addgene, plasmid# 87926) and pcDNA3.1-IDH2 R172K (deposited in Addgene, plasmid# 87927), IDH2 WT and IDH2 R172K were amplified by PCR from pCMV6-Entry vectors (Origene, Rockville, MD, cat# RC201152 and cat# RC400103) and cloned into the BamH1 and EcoR1 sites of the pcDNA3.1 expression vector (Invitrogen, Carlsbad, CA, cat# V790-20).

Techniques: Transfection, Mutagenesis, Plasmid Preparation, Purification, Gas Chromatography-Mass Spectrometry, Quantitation Assay, Whisker Assay, Comparison, Transformation Assay, Generated, Expressing, Control

Journal: eLife

Article Title: Replication Study: 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.26030

Figure Lengend Snippet: This is the same experiment as in , but with quantitation of 2HG signal intensity relative to glutamate determined using the EIC for each biological repeat. ( A ) Box and whisker plot with mean and 95% CI reported for each sample harvested 48 hr after transfection. R172K mutant IDH2 values were compared to the largest value observed in cells expressing vector control or wild-type IDH2 (0.004). One-sample t -test; t (9) = 22.4, uncorrected p =3.38×10 −9 , Bonferroni corrected p =6.77×10 −9 . ( B ) Representative EIC from samples harvested at 48 hr after transfection for vector control (left panels), wild-type IDH2 (middle panels), and R172K mutant IDH2 (right panels) with aspartate, glutamate, and 2HG shown. ( C ) Box and whisker plot with mean and 95% CI reported for each sample harvested 24 hr after transfection. R172K mutant IDH2 values were compared to the largest value observed in cells expressing vector control or wild-type IDH2 (0.001). One-sample Wilcoxon signed-rank test on log 10 transformed data; V = 55, uncorrected p =0.0020, Bonferroni corrected p =0.0039. ( D ) Representative EIC from samples harvested at 24 hr after transfection for vector control (left panels), wild-type IDH2 (middle panels), and R172K mutant IDH2 (right panels) with aspartate, glutamate, and 2HG shown. Additional details for this experiment can be found at https://osf.io/9ge2a/ . DOI: http://dx.doi.org/10.7554/eLife.26030.004

Article Snippet: To generate pcDNA3.1-IDH2 WT (deposited in Addgene, plasmid# 87926) and pcDNA3.1-IDH2 R172K (deposited in Addgene, plasmid# 87927), IDH2 WT and IDH2 R172K were amplified by PCR from pCMV6-Entry vectors (Origene, Rockville, MD, cat# RC201152 and cat# RC400103) and cloned into the BamH1 and EcoR1 sites of the pcDNA3.1 expression vector (Invitrogen, Carlsbad, CA, cat# V790-20).

Techniques: Quantitation Assay, Whisker Assay, Transfection, Mutagenesis, Expressing, Plasmid Preparation, Control, Transformation Assay

Journal: eLife

Article Title: Replication Study: 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.26030

Figure Lengend Snippet: AML patient peripheral blood or bone marrow samples were analyzed for intracellular metabolites. Cells had organic acids extracted, purified, and derivatized with MTBSTFA before analysis by GC-MS. Patient samples were prescreened for IDH genotypic status prior to metabolite analysis. ( A ) Quantitation of 2HG signal intensity relative to glutamate was determined using the TIC for each sample. Dot plot with means reported as crossbars. One-sample t -tests on log 10 transformed data comparing 2HG/glutamate level of samples to a constant of 0.0024 (2HG/glutamate threshold between IDH mutant and wild-type IDH samples). Samples with an IDH1 mutation compared to the threshold constant: t (3) = 4.47, uncorrected p =0.021, Bonferroni corrected p =0.063. Samples with an IDH2 mutation compared to the threshold constant: t (3) = 9.19, uncorrected p =0.0027, Bonferroni corrected p =0.0082. Samples with an IDH1 or IDH2 mutation compared to the threshold constant: t (7) = 8.73, uncorrected p =5.20×10 −5 , Bonferroni corrected p =1.56×10 −4 . ( B ) Representative TIC from samples without an IDH mutation (top panel), samples with an IDH1 mutation (middle panel), and samples with an IDH2 mutation (bottom panel). The derivatized organic acids eluting between 31.4 and 34.4 min are shown, including aspartate (31.7 min), 2HG (33.1 min), and glutamate (34.3 min) based on spectra of derivatized commercial standards. Additional details for this experiment can be found at https://osf.io/smdfr/ . DOI: http://dx.doi.org/10.7554/eLife.26030.005

Article Snippet: To generate pcDNA3.1-IDH2 WT (deposited in Addgene, plasmid# 87926) and pcDNA3.1-IDH2 R172K (deposited in Addgene, plasmid# 87927), IDH2 WT and IDH2 R172K were amplified by PCR from pCMV6-Entry vectors (Origene, Rockville, MD, cat# RC201152 and cat# RC400103) and cloned into the BamH1 and EcoR1 sites of the pcDNA3.1 expression vector (Invitrogen, Carlsbad, CA, cat# V790-20).

Techniques: Purification, Gas Chromatography-Mass Spectrometry, Quantitation Assay, Transformation Assay, Mutagenesis

Journal: eLife

Article Title: Replication Study: 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.26030

Figure Lengend Snippet: This is the same experiment as in , but with quantitation of 2HG signal intensity relative to glutamate determined using the EIC for each AML patient sample. ( A ) Dot plot with means reported as crossbars. One-sample t -tests on log 10 transformed data comparing 2HG/glutamate level of samples to a constant of 0.001 (2HG/glutamate threshold between IDH mutant and wild-type IDH samples). Samples with an IDH1 mutation compared to the threshold constant: t (3) = 5.40, uncorrected p =0.012, Bonferroni corrected p =0.037. Samples with an IDH2 mutation compared to the threshold constant: t (3) = 10.93, uncorrected p =0.0016, Bonferroni corrected p =0.0049. Samples with an IDH1 or IDH2 mutation compared to the threshold constant: t (7) = 10.44, uncorrected p =1.61×10 −5 , Bonferroni corrected p =4.84×10 −5 . ( B ) Representative EIC from samples without an IDH mutation (top panel), samples with an IDH1 mutation (middle panel), and samples with an IDH2 mutation (bottom panel) with aspartate, glutamate, and 2HG shown. Additional details for this experiment can be found at https://osf.io/smdfr/ . DOI: http://dx.doi.org/10.7554/eLife.26030.006

Article Snippet: To generate pcDNA3.1-IDH2 WT (deposited in Addgene, plasmid# 87926) and pcDNA3.1-IDH2 R172K (deposited in Addgene, plasmid# 87927), IDH2 WT and IDH2 R172K were amplified by PCR from pCMV6-Entry vectors (Origene, Rockville, MD, cat# RC201152 and cat# RC400103) and cloned into the BamH1 and EcoR1 sites of the pcDNA3.1 expression vector (Invitrogen, Carlsbad, CA, cat# V790-20).

Techniques: Quantitation Assay, Transformation Assay, Mutagenesis

Journal: eLife

Article Title: Replication Study: 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.26030

Figure Lengend Snippet: Effect size and 95% confidence interval are presented for , this replication study (RP:CB), and a random effects meta-analysis of those two effects. Sample sizes used in and this replication attempt are reported under the study name. Random effects meta-analysis of AML patient samples with an IDH1 mutation compared to a constant representing the 2HG/glutamate threshold between IDH mutant and wild-type IDH samples detected in the study (original: 0.01; replication: 0.0024) (meta-analysis p =0.114), AML patient samples with an IDH2 mutation compared to the threshold constant (meta-analysis p =1.12×10 −6 ), and AML patient samples with an IDH1 or IDH2 mutation compared to the threshold constant (meta-analysis p =5.73×10 −9 ). Additional details for these meta-analyses can be found at https://osf.io/4m3n8/ . DOI: http://dx.doi.org/10.7554/eLife.26030.007

Article Snippet: To generate pcDNA3.1-IDH2 WT (deposited in Addgene, plasmid# 87926) and pcDNA3.1-IDH2 R172K (deposited in Addgene, plasmid# 87927), IDH2 WT and IDH2 R172K were amplified by PCR from pCMV6-Entry vectors (Origene, Rockville, MD, cat# RC201152 and cat# RC400103) and cloned into the BamH1 and EcoR1 sites of the pcDNA3.1 expression vector (Invitrogen, Carlsbad, CA, cat# V790-20).

Techniques: Mutagenesis