Journal: American Journal of Cancer Research

Article Title: NDRG2 inhibits pyruvate carboxylase-mediated anaplerosis and combines with glutamine blockade to inhibit the proliferation of glioma cells

doi:

Figure Lengend Snippet: The expression of NDRG2 had an inverse association with pyruvate carboxylase in IDH1 (R132H)-mutant glioma cells. A. NDRG2 and pyruvate carboxylase immunostaining of tissue microarrays comprising IDH1 wild-type and IDH1 (R132H) glioma tissues with different differentiation states. Scale bar, 200 μm and 50 μm (magnification). B. Correlation analysis of the staining index for the expression of NDRG2 and PC proteins in the IDH1 wild-type glioma patient specimens (n = 27) and the IDH1-R132H mutant glioma patient specimens (n = 27). Pearson’s product-moment correlation coefficients and the P values are also shown. C. NDRG2 and pyruvate carboxylase expression in the IDH1 wild-type and IDH1 (R132H) glioma U87 cells. D. The relative protein levels of pyruvate carboxylase to β-actin were quantified by densitometry. *P<0.05, **P<0.01.

Article Snippet: The human glioma cell lines U251, T98G, IDH1 (R132H) U87, IDH1 (WT) U87 and HEK293T cells were purchased from ATCC and used in the present study.

Techniques: Expressing, Mutagenesis, Immunostaining, Staining

Journal: Translational oncology

Article Title: Wild-Type IDH1 and Mutant IDH1 Opposingly Regulate Podoplanin Expression in Glioma.

doi: 10.1016/j.tranon.2020.100758

Figure Lengend Snippet: Figure 1. The expression of Pdpn was significantly reduced in IDH-mutated glioma. (A) The mRNA levels of Pdpn were obtained and analyzed using TCGA LGG and GBM datasets. (B) The expression of Pdpn and IDH1 (R132H) was detected by IHC staining in astrocytoma (A), anaplastic astrocytoma (AA), and GBM with mutant (IDH-MU) and wild-type (IDH-WT) IDH. Bar = 100 μm. (C) The immunoreactivity of Pdpn in the specimens was scored: 0 (negative), 1 (weak), 2 (moderate), and 3 (strong staining) (D) The mRNA levels of Pdpn in IDH-MU and IDH-WT gliomas (n = 6 in each group) were analyzed by qPCR. *P < .05, **P < .01, ***P < .001. (E) Immunoblotting to detect Pdpn protein in IDH-MU and IDH-WT gliomas (A, AA, and GBM).

Article Snippet: To express the wild-type and mutant IDH1 in U87 and U251 cells, the wild-type IDH1 and mutant IDH1 (R132H) were subcloned into the pLenti-C-Myc-DDK-IRES-Puro vector (Origene) and pRRLSIN-cPPT-CMV- IRES-Hygro vector (Eureka Biotech, China), respectively, and the lentiviruses were generated and purified as previously described [29].

Techniques: Expressing, Immunohistochemistry, Mutagenesis, Staining, Western Blot

Journal: Translational oncology

Article Title: Wild-Type IDH1 and Mutant IDH1 Opposingly Regulate Podoplanin Expression in Glioma.

doi: 10.1016/j.tranon.2020.100758

Figure Lengend Snippet: Figure 3. Pdpn expression was remarkably upregulated in IDH-wild-type glioma and positively correlated with IDH1 levels. (A) TCGA datasets (http://www.cbioportal.org) showed that the IDH1 mRNA levels in IDH-wild-type GBM were higher than those in IDH-wild-type LGG, and both were correlated with Pdpn levels. (B) Correlation analysis was performed between total IDH1 (mutant and wild-type) and Pdpn mRNA levels in IDH-MU and IDH-WT gliomas. (C and D) IDH1 expression in IDH-MU and IDH-WT gliomas was analyzed by qPCR (C) and immunoblotting (D). (E) IHC staining showed that the total IDH1 (wild-type and mutant) was elevated in both IDH- wild-type (IDH-WT) and IDH-mutated (IDH-MU) GBM compared with the peritumor tissues, but the Pdpn protein levels were significantly increased only in IDH-WT GBM. Bar = 100 μm.

Article Snippet: To express the wild-type and mutant IDH1 in U87 and U251 cells, the wild-type IDH1 and mutant IDH1 (R132H) were subcloned into the pLenti-C-Myc-DDK-IRES-Puro vector (Origene) and pRRLSIN-cPPT-CMV- IRES-Hygro vector (Eureka Biotech, China), respectively, and the lentiviruses were generated and purified as previously described [29].

Techniques: Expressing, Mutagenesis, Western Blot, Immunohistochemistry

Journal: Translational oncology

Article Title: Wild-Type IDH1 and Mutant IDH1 Opposingly Regulate Podoplanin Expression in Glioma.

doi: 10.1016/j.tranon.2020.100758

Figure Lengend Snippet: Figure 4. Wild-type IDH1 greatly promoted Pdpn expression in glioma cells. Wild-type IDH1 (wtIDH1) was overexpressed in U251 and U87 glioma cells by lentivirus infection, and lentivirus with EV were used as controls. (A and B) The expression of IDH1 and Pdpn in U87 and U251 cells was analyzed by immunoblotting (A) and qPCR (n = 6) (B). (C) The human Pdpn promoter fragment (−857/+205) was cloned into the pGL3-basic vector, and the luciferase activity was determined in 293T cells with or without overexpressing wtIDH1. (D) Pdpn expression was measured in brain and primary glia of Idh1 knockout (Idh1LSL/LSL, −/−) and wild-type (+/+) mice. The results are representative of two or three independent experiments. The data are presented as the mean ± SE (n = 4). * P < .05, ** P < .01, and *** P < .001.

Article Snippet: To express the wild-type and mutant IDH1 in U87 and U251 cells, the wild-type IDH1 and mutant IDH1 (R132H) were subcloned into the pLenti-C-Myc-DDK-IRES-Puro vector (Origene) and pRRLSIN-cPPT-CMV- IRES-Hygro vector (Eureka Biotech, China), respectively, and the lentiviruses were generated and purified as previously described [29].

Techniques: Expressing, Infection, Western Blot, Clone Assay, Plasmid Preparation, Luciferase, Activity Assay, Knock-Out

Journal: Translational oncology

Article Title: Wild-Type IDH1 and Mutant IDH1 Opposingly Regulate Podoplanin Expression in Glioma.

doi: 10.1016/j.tranon.2020.100758

Figure Lengend Snippet: Figure 5. Mutant IDH1 and D-2-HG suppressed Pdpn expression in glioma cells. (A and B) Mutant IDH1 (muIDH1) was overexpressed in U87 and U251 cells by infection with lentivirus, individually or together with wild-type IDH1 (wtIDH1). Immunoblotting (A) and qPCR (B) detected the expression of IDH1, IDH1 (R132H), and Pdpn. (C) The transcriptional activity of the Pdpn promoter was analyzed by luciferase assay in 293T cells with wild-type and/or mutant IDH1 overexpression. (D and E) Primary glial cells were treated with different concentrations of Octyl-2-HG (cell permeable) or D-2-HG, and Pdpn expression was determined by immunoblotting (D) and qPCR (E). (F) U251 cells with wild-type IDH1 overexpression were treated with different concentrations of Octyl-2-HG (permeable) or D-2-HG, and Pdpn expression was determined by immunoblotting. (G) The transcriptional activities of the Pdpn promoter were determined in 293T cells with or without wild-type IDH1 by luciferase assay in the presence of Octyl-2-HG or D-2-HG. The results are representative of two or three independent experiments. The data are presented as the mean ± SE (n = 4). * P < .05, ** P < .01, and *** P < .001.

Article Snippet: To express the wild-type and mutant IDH1 in U87 and U251 cells, the wild-type IDH1 and mutant IDH1 (R132H) were subcloned into the pLenti-C-Myc-DDK-IRES-Puro vector (Origene) and pRRLSIN-cPPT-CMV- IRES-Hygro vector (Eureka Biotech, China), respectively, and the lentiviruses were generated and purified as previously described [29].

Techniques: Mutagenesis, Expressing, Infection, Western Blot, Activity Assay, Luciferase, Over Expression

Journal: Translational oncology

Article Title: Wild-Type IDH1 and Mutant IDH1 Opposingly Regulate Podoplanin Expression in Glioma.

doi: 10.1016/j.tranon.2020.100758

Figure Lengend Snippet: Figure 6. The methylation of the Pdpn promoter was opposingly regulated by wild-type and mutant IDH1 in glioma. (A) The methylation of 20 CpG islands in the Pdpn promoter was analyzed by bisulfite sequencing in IDH-MU and IDH-WT gliomas compared with peritumor tissues. (B) The methylation of CpG islands in Pdpn promoter was evaluated in U251 cells with overexpressing wild-type IDH1 (wtIDH1) and/or mutant IDH1 (muIDH1). (C) The levels of intracellular α-KG in IDH-MU and IDH-WT gliomas (n = 6). (D) The α-KG levels in primary glia of mutant IDH1 knockin mice (Idh1-KI) and control wild-type mice (Idh1-WT). (E) The α-KG levels in U251 glioma cells with overexpressing wtIDH1 and/or muIDH1 (n = 4). (F) U251 cells with overexpressed wtIDH1 were treated with different concentrations of Octyl-2-HG or D-2- HG, and the methylation of Pdpn promoter was determined by bisulfite sequencing. The results are representative of two or three independent experiments. The data are presented as the mean ± SE. * P < .05, ** P < .01, and *** P < .001.

Article Snippet: To express the wild-type and mutant IDH1 in U87 and U251 cells, the wild-type IDH1 and mutant IDH1 (R132H) were subcloned into the pLenti-C-Myc-DDK-IRES-Puro vector (Origene) and pRRLSIN-cPPT-CMV- IRES-Hygro vector (Eureka Biotech, China), respectively, and the lentiviruses were generated and purified as previously described [29].

Techniques: Methylation, Mutagenesis, Sequencing, Knock-In, Control

Journal: Translational oncology

Article Title: Wild-Type IDH1 and Mutant IDH1 Opposingly Regulate Podoplanin Expression in Glioma.

doi: 10.1016/j.tranon.2020.100758

Figure Lengend Snippet: Figure 7. The schematic diagram illustrated the interactive functions of wild-type and mutant IDH1 in glioma. The elevation of wild-type IDH1 (wtIDH1) in glioma cells hypomethylated the CpG island of Pdpn promoter by increasing the intracellular α-KG levels, while the mutant IDH (muIDH1) played an opposite function to reduce the intracellular α-KG levels and resulted in the hypermethylations of Pdpn promoter. D-2-HG, generated by muIDH1, also directly impeded the demethylation of Pdpn promoter at a higher concentration. The hypomethylation of Pdpn promoter increased the Pdpn transcription, which stimulated the migration of glioma cells.

Article Snippet: To express the wild-type and mutant IDH1 in U87 and U251 cells, the wild-type IDH1 and mutant IDH1 (R132H) were subcloned into the pLenti-C-Myc-DDK-IRES-Puro vector (Origene) and pRRLSIN-cPPT-CMV- IRES-Hygro vector (Eureka Biotech, China), respectively, and the lentiviruses were generated and purified as previously described [29].

Techniques: Mutagenesis, Generated, Concentration Assay, Migration

Journal: Oncotarget

Article Title: Oncogenic transgelin-2 is differentially regulated in isocitrate dehydrogenase wild-type vs. mutant gliomas

doi: 10.18632/oncotarget.26365

Figure Lengend Snippet: (A) TAGLN2 mRNA was expressed at significantly higher levels in IDH1/2 WT tumors compared to IDH1/2 mutant tumors in both our institutional (p-value=7.73×10 −5 ; FDR=0.053) and the validation TCGA (p-value <0.0001; FDR < 0.0001) cohorts. (B) Mass spectrometry identified higher TAGLN2 protein expression in IDH1/2 WT compared to IDH1/2 mutant LGG from our institutional cohort. Six different peptides corresponding to TAGLN2 protein were expressed at significantly up-regulated in IDH1/2 WT compared to IDH1/2 mutant tumors. (C) Publicly available TAGLN2 mRNA expression of all Grade II (G2, n=249), III (G3, n=265) and IV (GBM, n=153) gliomas from the TCGA database are significantly different (p<0.0001). (D) TAGLN2 expression in IDH1/2 WT Grade II (n=21), III (n=52) and IV (n=133) tumors from TCGA data was significantly different (p=5.136×10 −20 ).

Article Snippet: Human TAGLN2 (Myc-DDK-tagged) in pCMV6-entry vector (Origene, Rockville, MD) was transfected into GBM30, U87 MG IDH1/2 WT and U87 MG glioma cells overexpressing mutant IDH1R132H using Lipofectamine 2000 (Thermo Scientific, Waltham, MA) according to the manufacturer’s protocol.

Techniques: Mutagenesis, Biomarker Discovery, Mass Spectrometry, Expressing

Journal: Oncotarget

Article Title: Oncogenic transgelin-2 is differentially regulated in isocitrate dehydrogenase wild-type vs. mutant gliomas

doi: 10.18632/oncotarget.26365

Figure Lengend Snippet: (A) Promoter methylation was detected in IDH1/2 mutant tumors (n=54, cyan) and IDH1/2 WT tumors (n=8, salmon) from our institutional cohort using 15 CpG TAGLN2 promoter methylation sites included on the Illumina HM-450K array. IDH1/2 mutant showed significantly higher levels of methylation (FDR<0.05) in the majority of CpG islands corresponding to TAGLN2 (n=11), as demonstrated by the heat map. Low methylation levels are denoted in green and high methylation levels are denoted in red. (B) Methylation results were validated using methylation data from the publicly available TCGA cohort.

Article Snippet: Human TAGLN2 (Myc-DDK-tagged) in pCMV6-entry vector (Origene, Rockville, MD) was transfected into GBM30, U87 MG IDH1/2 WT and U87 MG glioma cells overexpressing mutant IDH1R132H using Lipofectamine 2000 (Thermo Scientific, Waltham, MA) according to the manufacturer’s protocol.

Techniques: Methylation, Mutagenesis

Journal: Oncotarget

Article Title: Oncogenic transgelin-2 is differentially regulated in isocitrate dehydrogenase wild-type vs. mutant gliomas

doi: 10.18632/oncotarget.26365

Figure Lengend Snippet: (A) TAGLN2 mRNA levels above the median (detected by Affymetrix Clariom D array) trended toward poor OS in lower grade gliomas from our institutional cohort (HR 3.74, p=0.079), as shown by the Kaplan Meier curve. (B) In the TCGA validation dataset, high TAGLN2 mRNA levels above the median were significantly associated with poor survival (HR= 2.6, p=0.00003).

Article Snippet: Human TAGLN2 (Myc-DDK-tagged) in pCMV6-entry vector (Origene, Rockville, MD) was transfected into GBM30, U87 MG IDH1/2 WT and U87 MG glioma cells overexpressing mutant IDH1R132H using Lipofectamine 2000 (Thermo Scientific, Waltham, MA) according to the manufacturer’s protocol.

Techniques: Biomarker Discovery

Journal: Oncotarget

Article Title: Oncogenic transgelin-2 is differentially regulated in isocitrate dehydrogenase wild-type vs. mutant gliomas

doi: 10.18632/oncotarget.26365

Figure Lengend Snippet: Clinical-pathological characteristics of patients analyzed for TAGLN2 mRNA expression in institutional and TCGA LGG cohorts

Article Snippet: Human TAGLN2 (Myc-DDK-tagged) in pCMV6-entry vector (Origene, Rockville, MD) was transfected into GBM30, U87 MG IDH1/2 WT and U87 MG glioma cells overexpressing mutant IDH1R132H using Lipofectamine 2000 (Thermo Scientific, Waltham, MA) according to the manufacturer’s protocol.

Techniques: Expressing, Mutagenesis

Journal: Oncotarget

Article Title: Oncogenic transgelin-2 is differentially regulated in isocitrate dehydrogenase wild-type vs. mutant gliomas

doi: 10.18632/oncotarget.26365

Figure Lengend Snippet: Multi-variable analysis of clinical-pathological factors with OS from low(er) grade gliomas in the TCGA cohort

Article Snippet: Human TAGLN2 (Myc-DDK-tagged) in pCMV6-entry vector (Origene, Rockville, MD) was transfected into GBM30, U87 MG IDH1/2 WT and U87 MG glioma cells overexpressing mutant IDH1R132H using Lipofectamine 2000 (Thermo Scientific, Waltham, MA) according to the manufacturer’s protocol.

Techniques: Biomarker Discovery

Journal: Oncotarget

Article Title: Oncogenic transgelin-2 is differentially regulated in isocitrate dehydrogenase wild-type vs. mutant gliomas

doi: 10.18632/oncotarget.26365

Figure Lengend Snippet: (A) GBM30 neurospheres stably expressing TAGLN2 shRNA and corresponding scrambled shRNA control were generated and the level of stable TAGLN2 knock-down detected by Western blot is shown. (B) GBM30 neurospheres with stable knock-down of TAGLN2 or scrambled shRNA control were counted at 24, 72, and 1120 hours after plating. Knock-down of TAGLN2 resulted in significantly decreased cell counts (p<0.05). (C) GBM30 neurospheres and (D) U87 MG glioma cells stably overexpressing TAGLN2 and corresponding vector control were generated and the level of stable TAGLN2 overexpression was detected by Western blot. Of note, endogenous TAGLN2 (22 Kda) and exogenous TAGLN2 -myc (28 kDa) are shown. (E) GBM30 neurospheres stably overexpressing TAGLN2 resulted in significantly increased cell proliferation compared to vector control at 72 and 120 hours (p<0.05). (F) U87 MG cells stably overexpressing TAGLN2 resulted in increased cell proliferation compared to the vector alone. Experiments were performed twice with six replicates each. * , statistically significant difference in proliferation.

Article Snippet: Human TAGLN2 (Myc-DDK-tagged) in pCMV6-entry vector (Origene, Rockville, MD) was transfected into GBM30, U87 MG IDH1/2 WT and U87 MG glioma cells overexpressing mutant IDH1R132H using Lipofectamine 2000 (Thermo Scientific, Waltham, MA) according to the manufacturer’s protocol.

Techniques: Stable Transfection, Expressing, shRNA, Control, Generated, Knockdown, Western Blot, Plasmid Preparation, Over Expression

Journal: Oncotarget

Article Title: Oncogenic transgelin-2 is differentially regulated in isocitrate dehydrogenase wild-type vs. mutant gliomas

doi: 10.18632/oncotarget.26365

Figure Lengend Snippet: Since TAGLN2 has been shown to play a role in invasion and metastases of other cancer types, the invasive ability of (A) GBM30 neurospheres with stable shRNA-mediated knock-down of TAGLN2 were compared to their respective scrambled shRNA control. GBM30 cells showed a decrease in average number of cells invading through the matrix after knock-down of TAGLN2 compared to control. In contrast, (B) GBM30 neurospheres and (C) U87 MG glioma cells with stable overexpression of TAGLN2 showed an increase in average number of cells invading through the matrix compared to vector control. Experiments were performed three times with triplicate invasion assays. * , statistically significant difference in invading cells (p<0.05). Photographs are representative images at 40x and 100x magnification.

Article Snippet: Human TAGLN2 (Myc-DDK-tagged) in pCMV6-entry vector (Origene, Rockville, MD) was transfected into GBM30, U87 MG IDH1/2 WT and U87 MG glioma cells overexpressing mutant IDH1R132H using Lipofectamine 2000 (Thermo Scientific, Waltham, MA) according to the manufacturer’s protocol.

Techniques: shRNA, Knockdown, Control, Over Expression, Plasmid Preparation

Journal: Oncotarget

Article Title: Oncogenic transgelin-2 is differentially regulated in isocitrate dehydrogenase wild-type vs. mutant gliomas

doi: 10.18632/oncotarget.26365

Figure Lengend Snippet: (A) TAGLN2 protein levels were compared in U87 MG IDH1/2 WT parental cells and a commercially available U87 MG isogenic cell line overexpressing IDH1 with a heterozygous R132H mutation by Western blot analysis. TAGLN2 protein was decreased in IDH1 mutant cells compared to IDH1/2 WT cells. (B) U87 MG IDH1 mutant cells were treated with increasing concentrations of 5-azacytidine (5-AZA) demethylating agent and TAGLN2 protein was evaluated by Western blot. 5-AZA resulted in increasing TAGLN2 protein levels expression in a dose-dependent manner.

Article Snippet: Human TAGLN2 (Myc-DDK-tagged) in pCMV6-entry vector (Origene, Rockville, MD) was transfected into GBM30, U87 MG IDH1/2 WT and U87 MG glioma cells overexpressing mutant IDH1R132H using Lipofectamine 2000 (Thermo Scientific, Waltham, MA) according to the manufacturer’s protocol.

Techniques: Mutagenesis, Western Blot, Expressing