Journal: Scientific reports

Article Title: Contribution of glucose and glutamine to hypoxia-induced lipid synthesis decreases, while contribution of acetate increases, during 3T3-L1 differentiation.

doi: 10.1038/s41598-024-79458-0

Figure Lengend Snippet: Fig. 3. Gene expression change of selected enzymes during adipocytes differentiation under hypoxia. Quantitative real-time PCR was employed to assess gene expression of (A_ pyruvate dehydrogenase E1 alpha 1 (PDHA1), (B) ATP citrate lyase (ACLY), and (C) acyl-CoA synthetase short-chain family member 2 (ACSS2), (D) isocitrate dehydrogenase 1 (IDH1), (E) isocitrate dehydrogenase 2 (IDH2). All values are expressed as 2^ΔCT. Data are presented as the mean ± SD, N = 6. Two-way ANOVA test was performed followed by Tukey’s post-hoc test.*p < 0.05 for comparison 4% O2 vs. 21% O2; #p < 0.05 for comparison 7 Days vs. 14 Days; ns = non-significant.

Article Snippet: Quantitative RT-PCR reactions were performed using the TaqMan probes (4331182, Applied Biosystems, USA) of Pdha1 (Mm00468675_m1), Acly (Mm01302282_m1), Acss2 (Mm00480101_m1), Idh1 (Mm00516030_m1), Idh2 (Mm00612429_m1), Gusb (Mm01197698_m1), Tbp (Mm01277042_m1) and Real Time PCR cycler ABI 750 (ThermoFisher Scientific, USA).

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Comparison

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: SUCLG2 Regulates Mitochondrial Dysfunction through Succinylation in Lung Adenocarcinoma.

doi: 10.1002/advs.202303535

Figure Lengend Snippet: Figure 3. SUCLG2 knockout induces a global increase in protein succinylation. A) Succinate levels in A549 and H1299 cells and SUCLG2 knockout stable cell lines. Data represent the average of three independent experiments (mean ± SD). ***p < 0.001. B) The succinylation of proteins was detected in A549-SUCLG2-KO and A549-WT cells using western blotting. C and D) The succinylation of proteins in A549-SUCLG2-KO and A549-WT cells was analyzed with a 4D mass spectrometer. Intracellular distribution of succinylated proteins (C). Biological process analysis showing that upregulated succinylated proteins were significantly enriched in processes related to mitochondrial dysfunction (D). n = 3 per group. E) Schematic depicting the key enzymes related to mitochondrial function by knock outing SUCLG2. F–J) The succinylation levels of GAPDH, ME2, IDH2, MDH2, and ACOT9 in A549-SUCLG2-KO and A549-WT cells measured by mass spectrometry. Data represent the average of three independent experiments (mean ± SD). *p < 0.05, **p < 0.01.

Article Snippet: The rabbit polyclonal antibodies against SUCLG2 (14240-1- AP, 1:2000), SUCLA2 (12627-1-AP, 1:1000), IDH2 (15932-1-AP, 1:1000), MDH2 (15462-1-AP, 1:1000), ME2 (24944-1-AP, 1:1000), and TRIM21 (12108-1-AP, 1:2000); the mouse monoclonal antibodies against β-tubulin (66240-1-Ig, 1:5000), SIRT5 (67257-1-Ig, 1:1000), ACOT9 (66532-1-lg, 1:5000), β-actin (60008-1-Ig, 1:4000), GAPDH (60004-1-Ig, 1:3000), the Flag tag (66008-4-Ig, 1:3000), and VDAC1 (66345-1-Ig, 1:2000) were ordered from Proteintech.

Techniques: Knock-Out, Stable Transfection, Western Blot, Mass Spectrometry

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: SUCLG2 Regulates Mitochondrial Dysfunction through Succinylation in Lung Adenocarcinoma.

doi: 10.1002/advs.202303535

Figure Lengend Snippet: Figure 4. SUCLG2 affects protein stability or enzymatic activity by regulating succinylation. A–E) Western blotting was used to detect the succinylation of the GAPDH, ME2, IDH2, MDH2, and ACOT9 proteins in A549-WT and A549-SUCLG2-KO cells. WCL: whole cell lysate. F–J) Western blotting was used to detect the succinylation of the GAPDH, ME2, IDH2, MDH2, and ACOT9 proteins in cells with or without SUCLG2 overexpression. K) The expression levels of GAPDH, ME2, IDH2, MDH2, and ACOT9 proteins in wild-type or SUCLG2 knockout stable cell lines were detected by western blotting. L) The cells were treated with 20 μg mL−1 CHX and collected at the indicated time. The degradation rate of the ME2 and ACOT9 proteins was detected by western blotting. M–P) The activity of GAPDH, ME2, IDH2, and MDH2 was detected using a specific enzyme activity detection kit. Data represent the average of three independent experiments (mean ± SD). ***p < 0.001.

Article Snippet: The rabbit polyclonal antibodies against SUCLG2 (14240-1- AP, 1:2000), SUCLA2 (12627-1-AP, 1:1000), IDH2 (15932-1-AP, 1:1000), MDH2 (15462-1-AP, 1:1000), ME2 (24944-1-AP, 1:1000), and TRIM21 (12108-1-AP, 1:2000); the mouse monoclonal antibodies against β-tubulin (66240-1-Ig, 1:5000), SIRT5 (67257-1-Ig, 1:1000), ACOT9 (66532-1-lg, 1:5000), β-actin (60008-1-Ig, 1:4000), GAPDH (60004-1-Ig, 1:3000), the Flag tag (66008-4-Ig, 1:3000), and VDAC1 (66345-1-Ig, 1:2000) were ordered from Proteintech.

Techniques: Activity Assay, Western Blot, Over Expression, Expressing, Knock-Out, Stable Transfection

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: Quantitative RT-PCR (qRT-PCR) 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: Quantitative RT-PCR (qRT-PCR) 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: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Discovery of novel enasidenib analogues targeting inhibition of mutant isocitrate dehydrogenase 2 as antileukaemic agents

doi: 10.1080/14756366.2022.2157411

Figure Lengend Snippet: Function of wild-type IDH in homeostasis and activity of mutant IDH in disease. α-KG, α-ketoglutarate; 2-HG, D-2-hydroxyglutarate; D2HGDH, D-2-hydroxyglutarate dehydrogenase; IDH, isocitrate dehydrogenase; IDH2m, mutant IDH2.

Article Snippet: Assay of IDH2 R140Q enzyme inhibition was performed using the fluorimetry-based assay according to manufacturer’s procedure of BPS Biosciences IDH2 (R140Q) Assay Kit, catalogue #79309 and wild type IDH Assay Kit, catalogue #71074.

Techniques: Activity Assay, Mutagenesis

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Discovery of novel enasidenib analogues targeting inhibition of mutant isocitrate dehydrogenase 2 as antileukaemic agents

doi: 10.1080/14756366.2022.2157411

Figure Lengend Snippet: Chemical structures of representative mutant IDH2 R140Q inhibitors.

Article Snippet: Assay of IDH2 R140Q enzyme inhibition was performed using the fluorimetry-based assay according to manufacturer’s procedure of BPS Biosciences IDH2 (R140Q) Assay Kit, catalogue #79309 and wild type IDH Assay Kit, catalogue #71074.

Techniques: Mutagenesis

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Discovery of novel enasidenib analogues targeting inhibition of mutant isocitrate dehydrogenase 2 as antileukaemic agents

doi: 10.1080/14756366.2022.2157411

Figure Lengend Snippet: In vitro inhibition of mutant and wild type IDH2 enzymes by selected target compounds.

Article Snippet: Assay of IDH2 R140Q enzyme inhibition was performed using the fluorimetry-based assay according to manufacturer’s procedure of BPS Biosciences IDH2 (R140Q) Assay Kit, catalogue #79309 and wild type IDH Assay Kit, catalogue #71074.

Techniques: In Vitro, Inhibition, Mutagenesis

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Discovery of novel enasidenib analogues targeting inhibition of mutant isocitrate dehydrogenase 2 as antileukaemic agents

doi: 10.1080/14756366.2022.2157411

Figure Lengend Snippet: Docking results and interacting residues for inhibitors, 6c , 6e , 7c , and enasidenib in IDH2 R140Q allosteric site (PDB ID: 5I96).

Article Snippet: Assay of IDH2 R140Q enzyme inhibition was performed using the fluorimetry-based assay according to manufacturer’s procedure of BPS Biosciences IDH2 (R140Q) Assay Kit, catalogue #79309 and wild type IDH Assay Kit, catalogue #71074.

Techniques:

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Discovery of novel enasidenib analogues targeting inhibition of mutant isocitrate dehydrogenase 2 as antileukaemic agents

doi: 10.1080/14756366.2022.2157411

Figure Lengend Snippet: Binding interaction of enasidenib (A) and compounds 6c (B), 6e (C), and 7c (D) inside IDH2 R140Q allosteric site (PDB ID: 5I96). 2D pose binding of the compound (left), green lines (H-bond), pink lines (hydrophobic interactions), cyan lines (halogen bond), and 3D surface representation of the compound in the allosteric site (right).

Article Snippet: Assay of IDH2 R140Q enzyme inhibition was performed using the fluorimetry-based assay according to manufacturer’s procedure of BPS Biosciences IDH2 (R140Q) Assay Kit, catalogue #79309 and wild type IDH Assay Kit, catalogue #71074.

Techniques: Binding Assay

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Discovery of novel enasidenib analogues targeting inhibition of mutant isocitrate dehydrogenase 2 as antileukaemic agents

doi: 10.1080/14756366.2022.2157411

Figure Lengend Snippet: Alignment of compounds 6c (dark red), 6e (blue), and 7c (orange) and enasidenib (green) in the IDH2 R140Q allosteric site viewing parallel layout.

Article Snippet: Assay of IDH2 R140Q enzyme inhibition was performed using the fluorimetry-based assay according to manufacturer’s procedure of BPS Biosciences IDH2 (R140Q) Assay Kit, catalogue #79309 and wild type IDH Assay Kit, catalogue #71074.

Techniques: