Journal: Amino Acids

Article Title: Arginine deprivation affects glioblastoma cell adhesion, invasiveness and actin cytoskeleton organization by impairment of β-actin arginylation

doi: 10.1007/s00726-014-1857-1

Figure Lengend Snippet: Effect of arginine deprivation on glioblastoma cells. a Cell growth of U251 ( upper panel ) and U87 ( lower panel ) cells cultivated in control, -Arg, -Lys and conditions. b U251 cell viability assessed under deprivation and re-supplementation conditions, as indicated. Upper and lower panels , arginine and lysine re-supplementation, respectively. 100 %, the number of the viable cells at time 0. Data in a and b are means ± SD; *** and *Statistical relevance p < 0.001 and p < 0.05, respectively

Article Snippet: Human glioblastoma U251 MG (U251) and U87 MG (U87) cell lines were purchased from CLS Cell Lines Service (Germany) and monitored for correct genetic profile via microsatellite analyses according to a protocol described earlier (Peickert et al. ).

Techniques:

Journal: Amino Acids

Article Title: Arginine deprivation affects glioblastoma cell adhesion, invasiveness and actin cytoskeleton organization by impairment of β-actin arginylation

doi: 10.1007/s00726-014-1857-1

Figure Lengend Snippet: Arginine deprivation affects morphology of glioblastoma but not glia cells. a , b , d Rat glia, U251 and U87 cells stained with Alexa 488-phalloidin, respectively. c Micrographs of U251 cells attained with scanning electron microscope. Insets in a , b and d ~2–3× magnification of the marked areas . Bars , in a , b and d 50 μm, and in c 10 μm. e U251 cells stained with Alexa 488-phalloidin before and after re-supplementation with Arg or Lys up to 0.4 and 0.8 mM concentration, respectively. Arrows point to lamellipodia, arrowheads point to elongated cells

Article Snippet: Human glioblastoma U251 MG (U251) and U87 MG (U87) cell lines were purchased from CLS Cell Lines Service (Germany) and monitored for correct genetic profile via microsatellite analyses according to a protocol described earlier (Peickert et al. ).

Techniques: Staining, Microscopy, Concentration Assay

Journal: Amino Acids

Article Title: Arginine deprivation affects glioblastoma cell adhesion, invasiveness and actin cytoskeleton organization by impairment of β-actin arginylation

doi: 10.1007/s00726-014-1857-1

Figure Lengend Snippet: Arginine deprivation impairs cell motility. a , b Migration tracks of U251 and U87 cells, respectively. Upper panels in a and b tracks of 10 randomly chosen cells; center panels images of migrating cells, and lower panels values of migration rate and mean distance based on tracks shown in upper panels . Values are means ± SD. ***Statistical relevance p < 0.001

Article Snippet: Human glioblastoma U251 MG (U251) and U87 MG (U87) cell lines were purchased from CLS Cell Lines Service (Germany) and monitored for correct genetic profile via microsatellite analyses according to a protocol described earlier (Peickert et al. ).

Techniques: Migration

Journal: Amino Acids

Article Title: Arginine deprivation affects glioblastoma cell adhesion, invasiveness and actin cytoskeleton organization by impairment of β-actin arginylation

doi: 10.1007/s00726-014-1857-1

Figure Lengend Snippet: Arginine deprivation impairs cell migration and invasiveness. Transwell filters not covered ( a ), and covered with Matrigel ( b , c) were used for analyses. Upper and lower panels in a and b images of U251 and U87 stained cells, respectively, taken on the filter trans side. c Images of LN-229 cells, analyzed as in b . Analyses were performed for three independent experiments run in duplicates. d Images of GFP-expressing U251 cells found within the E13 organotypic brain slice. The images represent the confocal 12.3-μm z -section of the planar center of brain slices. Right panel the quantification of GFP-expressing U251 cells within the confocal center of the slice per view area. The quantitative data in a – d are presented as % of control. Values are means ± SD. ***Statistical relevance p < 0.001

Article Snippet: Human glioblastoma U251 MG (U251) and U87 MG (U87) cell lines were purchased from CLS Cell Lines Service (Germany) and monitored for correct genetic profile via microsatellite analyses according to a protocol described earlier (Peickert et al. ).

Techniques: Migration, Staining, Expressing, Slice 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 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: Since TAGLN2 was found to be expressed at significantly lower mRNA and protein levels in IDH1/2 mutant gliomas from both institutional and TCGA patient cohorts, we confirmed these findings in vitro by evaluating TAGLN2 protein levels in a commercially available U87 MG isogenic cell line overexpressing IDH1 R132H mutant protein (ATCC, Manassas, VA), which will be referred to as IDH1 mutant U87 MG cells.

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: Since TAGLN2 was found to be expressed at significantly lower mRNA and protein levels in IDH1/2 mutant gliomas from both institutional and TCGA patient cohorts, we confirmed these findings in vitro by evaluating TAGLN2 protein levels in a commercially available U87 MG isogenic cell line overexpressing IDH1 R132H mutant protein (ATCC, Manassas, VA), which will be referred to as IDH1 mutant U87 MG cells.

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

Article Snippet: Since TAGLN2 was found to be expressed at significantly lower mRNA and protein levels in IDH1/2 mutant gliomas from both institutional and TCGA patient cohorts, we confirmed these findings in vitro by evaluating TAGLN2 protein levels in a commercially available U87 MG isogenic cell line overexpressing IDH1 R132H mutant protein (ATCC, Manassas, VA), which will be referred to as IDH1 mutant U87 MG cells.

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: Since TAGLN2 was found to be expressed at significantly lower mRNA and protein levels in IDH1/2 mutant gliomas from both institutional and TCGA patient cohorts, we confirmed these findings in vitro by evaluating TAGLN2 protein levels in a commercially available U87 MG isogenic cell line overexpressing IDH1 R132H mutant protein (ATCC, Manassas, VA), which will be referred to as IDH1 mutant U87 MG cells.

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: Since TAGLN2 was found to be expressed at significantly lower mRNA and protein levels in IDH1/2 mutant gliomas from both institutional and TCGA patient cohorts, we confirmed these findings in vitro by evaluating TAGLN2 protein levels in a commercially available U87 MG isogenic cell line overexpressing IDH1 R132H mutant protein (ATCC, Manassas, VA), which will be referred to as IDH1 mutant U87 MG cells.

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: Since TAGLN2 was found to be expressed at significantly lower mRNA and protein levels in IDH1/2 mutant gliomas from both institutional and TCGA patient cohorts, we confirmed these findings in vitro by evaluating TAGLN2 protein levels in a commercially available U87 MG isogenic cell line overexpressing IDH1 R132H mutant protein (ATCC, Manassas, VA), which will be referred to as IDH1 mutant U87 MG cells.

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: Since TAGLN2 was found to be expressed at significantly lower mRNA and protein levels in IDH1/2 mutant gliomas from both institutional and TCGA patient cohorts, we confirmed these findings in vitro by evaluating TAGLN2 protein levels in a commercially available U87 MG isogenic cell line overexpressing IDH1 R132H mutant protein (ATCC, Manassas, VA), which will be referred to as IDH1 mutant U87 MG cells.

Techniques: Mutagenesis, Western Blot, Expressing