pcdna3 1 entry vector  (Addgene inc)

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: 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