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: Journal of Cancer

Article Title: Preferential Association of Lissencephaly-1 Gene Expression with CD133+ Glioblastoma Cells

doi: 10.7150/jca.17635

Figure Lengend Snippet: Lis1 and CD133 gene expression in neurosphere-like U87 cells. (A) Exposure of U87 glioma cells to stem-conditioned medium (SM) induces phenotypic modifications resulting in neurospheres after five days, in both regular U87 cells (left) and shLis1-U87 cells (right). The expression of Lis1 (B) and CD133 (D) is induced in U87 cells exposed to SM, with the highest level at day 5 of incubation. (C) As expected, silencing Lis1 gene in U87 cells (shLis-U87) inhibits its induction in cells incubated with SM. CD133 induction is almost abrogated in shLis-U87 cells (E) compared with U87 cells (D). Lis1 is highly expressed in CD133+ cells isolated from U87 cell line and primary glioblastoma (HTC1 and HTC2) cell cultures in normal culture medium (CM) or SM (F) . Data show enrichment up to 60 fold in Lis1 expression in CD133 + fractions for cells grown in CM and up to 32 fold in cells incubated in SM. Lis1 expression in CD133+ fraction isolated from U87 cells grown in CM is 32 times higher than that of CD133- fraction (U87 columns); in CD133+ fraction isolated from U87 incubated in SM Lis1 expression is 35 times higher than in CD133 negative cells from the same culture. The negative control is represented by CD133+ cells isolated from shLis-U87 incubated in CM or SM for which Lis1 was not increased as compared with CD133- cells (shLis-U87/CM and SM columns).

Article Snippet: U87 cells were transfected with a mix of three plasmids, each containing specific shRNA for Lis1 using Plasmid Transfection Reagent in Plasmid Transfection Medium (Santa Cruz, CA).

Techniques: Gene Expression, Expressing, Incubation, Isolation, Negative Control

Journal: Journal of Cancer

Article Title: Preferential Association of Lissencephaly-1 Gene Expression with CD133+ Glioblastoma Cells

doi: 10.7150/jca.17635

Figure Lengend Snippet: Proliferation of irradiated U87 and shLis-U87 cells. Cells having Lis1 silenced or not were irradiated with X-ray doses from 5 to 50 Gy. Cells seeded at a density of 1x10 4 cells/well, in quadruplicates or triplicates in E-plates and placed in xCelligence RTCA instrument, were followed-up for 100 hours (A) . Alternatively, irradiated or not irradiated cells were seeded in 24-well plates and the DNA amount per well was determined using Hoechst 33342 (B) . Both methods showed that irradiated U87 cells recovered better their proliferative capacity than shLis-U87 cells.

Article Snippet: U87 cells were transfected with a mix of three plasmids, each containing specific shRNA for Lis1 using Plasmid Transfection Reagent in Plasmid Transfection Medium (Santa Cruz, CA).

Techniques: Irradiation

Journal: Journal of Cancer

Article Title: Preferential Association of Lissencephaly-1 Gene Expression with CD133+ Glioblastoma Cells

doi: 10.7150/jca.17635

Figure Lengend Snippet: Cell adhesion, migration and proliferation of CD133 + cells isolated from U87 and shLis-U87 cells . CD133+ cells were isolated from control U87 and shLis-U87 cells. The purity of the fraction is revealed by higher CD133 expression (assessed by RT-PCR) in CD133+ fraction as compared with CD133- fraction (A) . CD133+ cells isolated from control U87 (blue circles) or from shLis-U87 (red squares) cultures were subjected to functional assays using xCELLigence Real-Time Cell Analysis instruments. The data representing the recorded cell index at different times show that CD133+ cells isolated from shLis-U87 culture present two times lower (B) adherence to the surface, (C) migratory potential and (D) proliferative rate, as compared with the those isolated from control U87 culture.

Article Snippet: U87 cells were transfected with a mix of three plasmids, each containing specific shRNA for Lis1 using Plasmid Transfection Reagent in Plasmid Transfection Medium (Santa Cruz, CA).

Techniques: Migration, Isolation, Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Functional Assay, Cell Analysis

Journal: Nanoscale Research Letters

Article Title: Examining the Roles of Emulsion Droplet Size and Surfactant in the Interfacial Instability-Based Fabrication Process of Micellar Nanocrystals

doi: 10.1186/s11671-017-2202-x

Figure Lengend Snippet: Interactions of PS-PEG micellar QDs (prepared by using sonication 30 s in the interfacial instability method) with biological cells. a PS-PEG micellar QDs were fairly biocompatible judging from the MTT cytotoxicity assay results. The concentrations were based on the amounts of QDs used. b Tat peptide-conjugated PS-PEG micellar QDs can be internalized by live cells. c RGD peptide-conjugated PS-PEG micellar QDs can specifically recognize and bind with the α v β 3 -integrin molecules over-expressed on U87MG cells ( right image ). In comparison, in the absence of α v β 3 -integrin over-expression (MCF-7 cells, left image ) or Tat peptide (PS-PEG-COOH micellar QDs, middle image ), no significant binding (QD fluorescence) was observed. The red fluorescence was from QDs. The cell nucleus was stained by the blue fluorescent dye Hoechst 33342. Cell periphery is shown by white line (from the corresponding bright field microscopy images)

Article Snippet: To counter-stain the cell nucleus, right before imaging (at a particular time point of cellular transport), the fluorescent dye Hoechst 33342 (5 μM in cell culture medium) was incubated with live cells for 20 min. Live cell imaging was also applied to study the specific binding of RGD peptide-conjugated PS-PEG micellar QDs with α v β 3 -integrin molecules, using an α v β 3 -integrin over-expressed cell line (U87 MG cells, purchased from KeyGen Biotech, China) versus a cell line without α v β 3 -integrin over-expression (MCF-7 cells, purchased from KeyGen Biotech, China).

Techniques: Sonication, Cytotoxicity Assay, Comparison, Over Expression, Binding Assay, Fluorescence, Staining, Microscopy

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