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

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: BMC Genomics

Article Title: Hypoxic signature of microRNAs in glioblastoma: insights from small RNA deep sequencing

doi: 10.1186/1471-2164-15-686

Figure Lengend Snippet: Hypoxia regulated miRNAs. Hierarchical clustering of hypoxia-induced and down-regulated miRNAs (>1.5-fold) in response to hypoxia (0.2% O 2 ) in cell line U87MG (a) . List of hypoxia-regulated miRNA clusters in U87MG cells (b) . A table showing correlation of microRNAs altered in hypoxia or in GBM tumor tissues (c) .

Article Snippet: Cell line U87MG was obtained from the National Centre for Cell Sciences, Pune.

Techniques:

Journal: BMC Genomics

Article Title: Hypoxic signature of microRNAs in glioblastoma: insights from small RNA deep sequencing

doi: 10.1186/1471-2164-15-686

Figure Lengend Snippet: Quantitative RT-PCR data showing miRNA levels in response to hypoxia or HIF1A. Graph showing miRNAs that are upregulated (a) or downregulated (b) in response to hypoxia. (c) U87MG cells were transfected with pCDNA3.1or a HIF1A over-expressing plasmid (pCDNA3.1-HIF1A), and miRNA levels were determined. U87MG cells were transfected with pLK0.1-shGFP or a shHIF1A over-expressing plasmid (pLK0.1-shHIF1A) and levels of HIF1A (d) and miRNAs (e) in response to hypoxia were determined. The graphical data points represent mean ± S.D. of at least three independent experiments. (*P > 0.01 and < 0.05; **P < 0.01). Error bars denote ± S. D.

Article Snippet: Cell line U87MG was obtained from the National Centre for Cell Sciences, Pune.

Techniques: Quantitative RT-PCR, Transfection, Expressing, Plasmid Preparation

Journal: BMC Genomics

Article Title: Hypoxic signature of microRNAs in glioblastoma: insights from small RNA deep sequencing

doi: 10.1186/1471-2164-15-686

Figure Lengend Snippet: MiR-210-3p induces HIF transcriptional activity. U87MG cells were transfected with a HRE luciferase vector, along with either miR-210-3p overexpression - [(pBABE-miR210) or control (pBABE)] or inhibition - [(miR-210 inhibitor) or (control)] vectors, and HRE transcriptional activity was assayed (a) . U87MG cells were transiently transfected with either a miR-210-3p over-expression vector (pBABE-miR-210) or the empty pBABE-puro parent vector (b) or with either a miR-210-3p inhibitor or control oligos (c) , and VEGF/CA9 levels were determined by qRT-PCR. The graphical data points represent mean ± S.D. of at least three independent experiments. (*P > 0.01 and < 0.05; **P < 0.01). Error bars denote ± S. D.

Article Snippet: Cell line U87MG was obtained from the National Centre for Cell Sciences, Pune.

Techniques: Activity Assay, Transfection, Luciferase, Plasmid Preparation, Over Expression, Control, Inhibition, Quantitative RT-PCR

Journal: BMC Genomics

Article Title: Hypoxic signature of microRNAs in glioblastoma: insights from small RNA deep sequencing

doi: 10.1186/1471-2164-15-686

Figure Lengend Snippet: MiR-210-3p functions. Graphs showing MTT assay results of cell survival on the 3 rd day in U87MG, U251MG and A172 cells in response to either miR-210 overexpression - [miR-210 polyclonals (pBABE-miR-210) or control (pBABE)] or inhibition - [(miR-210 inhibitor) or (control)] under (a) 0.2% hypoxia (b) or serum starvation or (c) chemo drug temozolomide treatment. The graphical data points represent mean ± S.D. of at least three independent experiments. (*P > 0.01 and < 0.05; **P < 0.01). Error bars denote ± S. D.

Article Snippet: Cell line U87MG was obtained from the National Centre for Cell Sciences, Pune.

Techniques: MTT Assay, Over Expression, Control, Inhibition

Journal: iScience

Article Title: Cancer cell intrinsic TIM-3 induces glioblastoma progression

doi: 10.1016/j.isci.2022.105329

Figure Lengend Snippet: TIM-3 is one of the most highly elevated immune checkpoints in GBM and is expressed by both non-tumor and tumor cells (A) The expression analyses of immune checkpoints in GBM, in comparison with non-tumor tissue and LGG, identified 7 overlapping elevated immune checkpoints, including 2 co-inhibitory immune checkpoints ( TIM- 3 and LAIR1 ) and 5 co-stimulatory immune checkpoints ( SLAMF8 , CD300A , TYPOBP , CD58 , and BTN3A2 ) (TCGA-seq, GBM, n = 155; LGG, n = 515; non-tumor, n = 4; GSE16011 , GBM, n = 155; LGG, n = 116; non-tumor, n = 8). (B) RT-qPCR analyses of CTLA4 , TIM3 , LAIR1 , PD1 , PDL1 , PDL2 and ID O 1 in clinical GBM samples (n = 10, means ± SEM, one-way ANOVA). (C) Immunohistochemical staining (Left, scale bar, 50 μm) and analyses (Right, non-tumor, n = 13; Grade II, n = 6; Grade III, n = 17; Grade IV, n = 77; means ± SEM, one-way ANOVA) of TIM-3 in clinical samples. (D) The Kaplan-Meier analyses of clinical glioma samples reveal the correlation of TIM-3 with poor prognosis in GBM (TIM-3 high vs. low, p = 0.0003; log rank test). (E) CIBERSORT analysis of non-tumor cell populations associated with high TIM-3 expression in TCGA RNA-seq GBM dataset (Pearson correlation analysis). (F) The tSNE plot of TIM-3 expression profile in GBM and immune cells with single-cell RNA-seq data ( GSE131928 ). (G) Representative immunofluorescence images of TIM-3 and GFAP staining in clinical GBM samples (scale bar, 50 μm). (H) FACS analysis shows that clinical GBM samples contained low CD45, and high TIM-3 cell populations (n = 7). (I) qPCR analysis of TIM-3 mRNA expression in NHA, indicated glioma cell lines (U87, U251, and LN229), primary glioma cells (PGC1228, PGC21, PGC24, and PGC40), and PBMC from patients with glioma (G-PBMC1 and G-PBMC2) and healthy donor (PBMC1 and PBMC2) (n = 3, means ± SEM, one-way ANOVA). (J) Western blot analyses of TIM-3 in NHA, indicated glioma cell lines, and primary glioma adherent and neurosphere cells (n = 3, means ± SEM, one-way ANOVA). (K) The schematic diagram describing the co-culture system of THP-1-derived anti-inflammatory/pro-tumorigenic (M2) macrophages and glioma cells (left panel), and representative western blot images of TIM-3 in indicated glioma cells cocultured with THP-1-derived anti-inflammatory/pro-tumorigenic TAMs (right panel). (ns p ≥ 0.05, ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗∗ p < 0.0001).

Article Snippet: Human: U87 cells , GeneChem , RRID: CVCL_0022.

Techniques: Expressing, Comparison, Quantitative RT-PCR, Immunohistochemical staining, Staining, RNA Sequencing, Immunofluorescence, Western Blot, Co-Culture Assay, Derivative Assay

Journal: iScience

Article Title: Cancer cell intrinsic TIM-3 induces glioblastoma progression

doi: 10.1016/j.isci.2022.105329

Figure Lengend Snippet: TIM-3 increases IL6 expression via activating NF-κB signaling in glioma cells (A) Representative western blotting images of Gal-9 in conditioned medium (CM) from indicated glioma cells separately transfected with TIM-3 overexpression, control, knockdown, or siNC vector. (B) The detection (left) and quantification (right) of cytokines in culture supernatants from GSC40 transduced with TIM-3 overexpression or control vector by proteome profiler cytokine array. (C-E) Representative western blotting images of IL6 in U87 cells transduced with control (U87-NC) or TIM-3 overexpression vectors (U87-TIM3 OE), respectively, and then with indicated treatment at indicated incubation time (C, with CHX; D, with CHX after MG132 pretreatment; E, with CHX after CQ pretreatment). (F) Representative western blot images of indicated glioma cells transduced with TIM-3 overexpression vector with or without blockade of Gal-9 (10 μg/mL). (G) IL6 concentration in CM from indicated cells measured by ELISA (n = 3, means ± SEM, one-way ANOVA). (H) Representative western blot images of indicated cells incubated with indicated concentrations (ng/mL) of IL6. (I-K) The decreased growth (I, n = 3, means ± SEM, one-way ANOVA; scale bar, 50 μm), migration (J, n = 3, means ± SEM, one-way ANOVA; scale bar, 50 μm), and neurosphere formation abilities (K, upper: n = 10, extreme limiting dilution assay; lower: stem cell frequency, n = 3, means ± SEM, one-way ANOVA) of GSC40 induced by TIM-3 knockdown was partially rescued by IL6 supplement (40 ng/mL). (ns p ≥ 0.05; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001).

Article Snippet: Human: U87 cells , GeneChem , RRID: CVCL_0022.

Techniques: Expressing, Western Blot, Transfection, Over Expression, Control, Knockdown, Plasmid Preparation, Transduction, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Migration, Limiting Dilution Assay

Journal: iScience

Article Title: Cancer cell intrinsic TIM-3 induces glioblastoma progression

doi: 10.1016/j.isci.2022.105329

Figure Lengend Snippet:

Article Snippet: Human: U87 cells , GeneChem , RRID: CVCL_0022.

Techniques: Control, Purification, Recombinant, Modification, Sterility, Cell Culture, Lysis, Transfection, Western Blot, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Proliferation Assay, Imaging, Sequencing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Software, Fluorescence, Microscopy