Journal: Cancer Science

Article Title: RBPJ contributes to the malignancy of glioblastoma and induction of proneural‐mesenchymal transition via IL‐6‐STAT3 pathway

doi: 10.1111/cas.14642

Figure Lengend Snippet: Effects of RBPJ knockdown on the proliferation and invasiveness of glioblastoma (GBM) cells. A, Expression of RBPJ in 2 GBM cell lines (U251 and T98), as evaluated by western blotting. HEK293 cells served as a positive control for RBPJ expression. B, Decreased RBPJ protein level in cells transduced with both RBPJ‐targeting small hairpin RNA (shRNA)1 and 2 compared with the control in GBM cells. C, Proliferation assay using Alamar blue. Both shRBPJ1 and 2 suppressed proliferation ability of GBM cells. Data are presented as the mean ± standard deviation (SD) (n = 6). * P < .05; ** P < .01, *** P < .001. D, Invasiveness of RBPJ‐silenced and control GBM cells, as assessed by transwell assay. Invasiveness of RBPJ‐silenced GBM cells was reduced compared with that of control cells. The bars represent mean cell counts from at least 6 high‐powered fields (HPF). * P < .05; ** P < .01, *** P < .001

Article Snippet: HEK293 cells and human GBM cell lines U251 and T98 were purchased from the American Type Culture Collection.

Techniques: Knockdown, Expressing, Western Blot, Positive Control, Transduction, shRNA, Control, Proliferation Assay, Standard Deviation, Transwell Assay

Journal: CNS Neuroscience & Therapeutics

Article Title: Curcumin Suppresses Malignant Glioma Cells Growth and Induces Apoptosis by Inhibition of SHH / GLI 1 Signaling Pathway in Vitro and Vivo

doi: 10.1111/cns.12163

Figure Lengend Snippet: Cytotoxic effect of curcumin on human glioma cells. (A) Chemical structure and molecular weight of curcumin. (B) U87 and T98G cells were cultured in‐96‐well plates, respectively, and treated with increasing concentrations of curcumin for 24 h. Cell death was analyzed using MTT assay. The error bars represent standard error. Data are shown as mean ± SEM for the three replicates. Statistical significance levels are indicated as: *P < 0.05; **P < 0.01 and ***P < 0.001.

Article Snippet: Western Blot Analysis Treated and untreated U87 and T98G cells were scraped in Thermo scientific RIPA buffer (Pierce, USA) with protease inhibitors.

Techniques: Molecular Weight, Cell Culture, MTT Assay

Journal: CNS Neuroscience & Therapeutics

Article Title: Curcumin Suppresses Malignant Glioma Cells Growth and Induces Apoptosis by Inhibition of SHH / GLI 1 Signaling Pathway in Vitro and Vivo

doi: 10.1111/cns.12163

Figure Lengend Snippet: Curcumin suppressed proliferation of U87 and T98G cells. (A) Cells were plated and grown for 24 h and then treated with solvent only (Con) or increasing doses of curcumin as indicated and harvested after 24, 48, and 72 h. Cell viability was examined using the MTT assay. Curcumin treatment resulted in significant decrease dose‐ and time‐dependent manner in cell proliferation in both two cell lines when compared with untreated controls. (B) Curcumin inhibited colony formation. Human glioma cells were incubated with indicated concentrations of curcumin for 24 h and allowed to grow into colonies for 12 days. Incubation with curcumin inhibits colony formation. Data are mean ± SEM for the three replicates. *P < 0.05; **P < 0.01 and ***P < 0.001.

Article Snippet: Western Blot Analysis Treated and untreated U87 and T98G cells were scraped in Thermo scientific RIPA buffer (Pierce, USA) with protease inhibitors.

Techniques: Solvent, MTT Assay, Incubation

Journal: CNS Neuroscience & Therapeutics

Article Title: Curcumin Suppresses Malignant Glioma Cells Growth and Induces Apoptosis by Inhibition of SHH / GLI 1 Signaling Pathway in Vitro and Vivo

doi: 10.1111/cns.12163

Figure Lengend Snippet: Curcumin treatment decreased GLI1 expression at the cytoplasma,nucleus and inactivated consensus GLI1‐binding sites. (A) T98G cells treated with 20 uM of curcumin for 24 h were subjected to immunofluorescent staining using anti‐GLI1 antibody. Curcumin treatment prevented most of GLI1 translocation into nucleus and resulted in lower expression levels at the cytoplasma. Bars represent 20 um. (B) U87 and T98G cells were transiently transfected (24 h) with a firefly luciferase reporter construct containing eight consecutive consensus GLI1‐binding sites (8×‐GLI1) and cotransfected with pGL4.75. Cells were then treated as indicated with solvent DMSO (control) or curcumin. Both firefly and Renilla luciferase activities were quantified, and the firefly/Renilla luciferase activities were recorded as fold induction over solvent‐treated (Con) cells transfected with the 8×‐GLI1/pGL4.75 vector reporter constructs. Mean ± SEM (n = 3; *P < 0.05).

Article Snippet: Western Blot Analysis Treated and untreated U87 and T98G cells were scraped in Thermo scientific RIPA buffer (Pierce, USA) with protease inhibitors.

Techniques: Expressing, Binding Assay, Staining, Translocation Assay, Transfection, Luciferase, Construct, Solvent, Control, Plasmid Preparation

Journal: CNS Neuroscience & Therapeutics

Article Title: Curcumin Suppresses Malignant Glioma Cells Growth and Induces Apoptosis by Inhibition of SHH / GLI 1 Signaling Pathway in Vitro and Vivo

doi: 10.1111/cns.12163

Figure Lengend Snippet: Curcumin triggered apoptosis through the internal pathway in glioma cells. (A) Subconfluent cells were either solvent DMSO‐treated (Con) or challenged with the indicated concentrations of curcumin for 24 h, and then, cell apoptosis was examined by flow cytometry assay using the Annexin V/PI. A representative experiment of the three replicated assays is shown. (B) Histogram representing dose‐dependent apoptosis of the U87 and T98G cell lines. Data are mean ± SEM of three replicates. *P < 0.05; **P < 0.01; ***P < 0.001. (C) The T98G cells were treated with 40 uM curcumin for the indicated periods of time, and then, cell extracts were prepared and used for Western blot assay using the indicated antibodies. Results showed that Bcl‐2 decreased following the increasing time points. Others were increased in a time‐dependent style. (D) Graph showing the Bax/Bcl‐2 ratio. Error bars represent standard error of three different experiments.

Article Snippet: Western Blot Analysis Treated and untreated U87 and T98G cells were scraped in Thermo scientific RIPA buffer (Pierce, USA) with protease inhibitors.

Techniques: Solvent, Flow Cytometry, Western Blot

Journal: CNS Neuroscience & Therapeutics

Article Title: Curcumin Suppresses Malignant Glioma Cells Growth and Induces Apoptosis by Inhibition of SHH / GLI 1 Signaling Pathway in Vitro and Vivo

doi: 10.1111/cns.12163

Figure Lengend Snippet: Curcumin inhibited SHH/GLI1 signaling and its downstream targets oncoproteins in time‐ and dose‐dependent manner. (A) Real‐time everse transcription‐PCR analysis of total RNA from T98G cells following 40 uM of curcumin treatment for different time points (0, 8, 16, 24 h) revealed decreasing expression of Shh, Smo, and GLI1 on mRNA level. (B) Western blot assay showed curcumin treatment led to decreased levels of Shh, GLI1, and GLI1‐regulated target (CyclinD1, Bcl‐2, Foxm1) proteins in time‐dependent manner in T98G cells following 40 uM agent exposure for 24 h. (C) U87 and T98G cells were plated and grown for 24 h and then treated with the solvent DMSO (control) or with different (indicated) doses of curcumin dissolved in DMSO for 24 h before harvesting. Western blot assay shows curcumin inhibits core components of the SHH/GLI1 signaling (SHH, GLI1) and its target oncoproteins (CyclinD1, Bcl‐2, Foxm1) in dose‐dependent manner. Data are shown as mean ± SEM for the three replicates. Statistical significance levels are indicated as: *P < 0.05; **P < 0.01 and ***P < 0.001.

Article Snippet: Western Blot Analysis Treated and untreated U87 and T98G cells were scraped in Thermo scientific RIPA buffer (Pierce, USA) with protease inhibitors.

Techniques: Expressing, Western Blot, Solvent, Control

Journal: BMC Biology

Article Title: Targeting of REST with rationally-designed small molecule compounds exhibits synergetic therapeutic potential in human glioblastoma cells

doi: 10.1186/s12915-024-01879-0

Figure Lengend Snippet: REST promotes glioblastoma growth. A Boxplot of REST mRNA expression in TCGA-LGG and TCGA-GBM samples compared to normal brain samples from TCGA and GTEx datasets (* p < 0.001). B Survival analysis using data from TCGA-GBM and TCGA-LGG projects. Analysis was done using GEPIA2 web server ( A , B ). C Basal REST protein amount in a panel of select cell lines. Three to four independent biological replicates are shown as mean ± SD. Statistical difference vs SVGp12 was tested using ANOVA with post hoc tests. Individual data values are provided in Additional File A. D Proliferation of GBM cell lines assessed by counting cells every 24 h. Shown is one representative replicate and quantification of PDT based on three independent experiments (mean ± SD). Statistical difference vs U251 was tested using ANOVA with post hoc tests. Individual data values are provided in Additional File B. E Western blot confirms the lack of REST in homozygous REST-KO clones of T98G ( left ) and HEK293 ( right ). F Proliferation of WT and REST-KO T98G cells was examined by counting cells every 24 h. Shown is one representative replicate and quantification of PDT based on three to four independent experiments (mean ± SD). Statistical comparison vs T98G control was performed using ANOVA with post hoc tests. Individual data values are provided in Additional File C. G Proliferation of T98G WT and REST-KO C10 cells (transfected with empty vector pLPC vs REST OE) was examined by counting cells every 24 h. Shown is one representative replicate and quantification of PDT based on three independent experiments. Statistical difference was tested using two-tailed paired t -test. Individual data values are provided in Additional File D. H Wound scratch assay and its quantification using ImageJ. Shown are mean ± SD from three independent biological experiments. Groups were compared using paired t -tests. Individual data values are provided in Additional File E. I Effect of REST loss on GSC marker expression. Shown are fold changes (FC) vs CRISPR Control derived from three independent biological replicates. Comparison vs control was performed using unpaired one-tailed t -tests. Dashed line indicates FC = 1. Individual data values are provided in Additional File F. *** p < 0.001; ** p < 0.01; * p < 0.05; ns—not significant

Article Snippet: Human embryonic kidney cells (HEK293/HEK293T), human liver cancer cells (HepG2), human fetal glial cells SVGp12, and glioblastoma cell lines (A172, T98G) were purchased from ATCC (Manassas, VA).

Techniques: Expressing, Western Blot, Clone Assay, Comparison, Control, Transfection, Plasmid Preparation, Two Tailed Test, Wound Healing Assay, Marker, CRISPR, Derivative Assay, One-tailed Test

Journal: BMC Biology

Article Title: Targeting of REST with rationally-designed small molecule compounds exhibits synergetic therapeutic potential in human glioblastoma cells

doi: 10.1186/s12915-024-01879-0

Figure Lengend Snippet: Transcriptome sequencing reveals distinct changes in gene signatures associated with REST. A RNA-seq analyses showing gene upregulation and downregulation (highlighted in red, log 2 FC cutoff = 0.58, p adj < 0.05) in REST KO cells compared to corresponding control (T98G— left , HEK293— right ). Representative volcano plots were built using “Enhanced Volcano” Bioconductor package. B Venn diagrams of deregulated genes shared between three “slow” T98G REST-KO clones (C10, F7, and G2): upregulated genes are shown on the left, downregulated genes are shown on the right. C GO (gene ontology) categories enriched among upregulated genes ( top ) and downregulated genes ( bottom ) in T98G REST-KO vs control. D Overlap of upregulated genes in glioblastoma ( left , shared between three “slow” clones, in green) and HEK293 ( right , shared between two clones, in green) and a published subset of genes with REST binding sites in human embryonic stem cells (in yellow) . E A list of representative REST-target genes ( n = 6) based on Tag-Seq and TCGA-GBM data analysis (shown are correlation coefficients of gene expression with REST mRNA). F Validation of REST-target genes by qPCR assay in REST-KO cells. Shown are fold changes (FC) vs T98G control cells derived from three independent biological replicates. Gene expression was measured using ddCt method and normalized by ACTB expression. Comparison vs control was performed using one-tailed t -tests. Dashed line indicates FC = 1.5. Individual data values are provided in Additional File G

Article Snippet: Human embryonic kidney cells (HEK293/HEK293T), human liver cancer cells (HepG2), human fetal glial cells SVGp12, and glioblastoma cell lines (A172, T98G) were purchased from ATCC (Manassas, VA).

Techniques: Sequencing, RNA Sequencing, Control, Clone Assay, Binding Assay, Gene Expression, Biomarker Discovery, Derivative Assay, Expressing, Comparison, One-tailed Test

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Sulphonamide derivatives with a triazine core as novel inducers of apoptosis and pyroptosis in glioblastoma multiforme cells

doi: 10.1080/14756366.2025.2594158

Figure Lengend Snippet: Results of LDH measurement of A172 (A) and U-87 MG (B) glioblastoma cells after 24 h of incubation with compounds. Means ± SD are shown with N = 3. ANOVA tests were used to demonstrate differences between cells treated with compounds and control. ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001.

Article Snippet: Human glioblastoma cell lines (A172 CRL-1620 TM , U-87 MG HTB-14 TM , U-118 MG HTB-15 TM , T98G CRL-1690 TM ) and the mouse astrocyte cell line (C8-D1A CRL-2541 TM ) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Incubation, Control

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Sulphonamide derivatives with a triazine core as novel inducers of apoptosis and pyroptosis in glioblastoma multiforme cells

doi: 10.1080/14756366.2025.2594158

Figure Lengend Snippet: Results of flow cytometry analysis of A172 (A) and U-87 MG (B) glioblastoma cells after 24 h of incubation with compounds. Means ± SD are shown with N = 4.

Article Snippet: Human glioblastoma cell lines (A172 CRL-1620 TM , U-87 MG HTB-14 TM , U-118 MG HTB-15 TM , T98G CRL-1690 TM ) and the mouse astrocyte cell line (C8-D1A CRL-2541 TM ) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Flow Cytometry, Incubation

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Sulphonamide derivatives with a triazine core as novel inducers of apoptosis and pyroptosis in glioblastoma multiforme cells

doi: 10.1080/14756366.2025.2594158

Figure Lengend Snippet: Results of flow cytometry analysis of A172 (A) and U-87 MG (B) glioblastoma cells after 24 h of incubation with compounds. Graphs visualise the cytograms from the compound analysis A172 (C) and U-87 MG (D). Means ± SD are shown with N = 4. ANOVA tests were used to demonstrate differences between cells treated with compounds and control. *** P ≤ 0.001; **** P ≤ 0.0001.

Article Snippet: Human glioblastoma cell lines (A172 CRL-1620 TM , U-87 MG HTB-14 TM , U-118 MG HTB-15 TM , T98G CRL-1690 TM ) and the mouse astrocyte cell line (C8-D1A CRL-2541 TM ) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Flow Cytometry, Incubation, Control

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Sulphonamide derivatives with a triazine core as novel inducers of apoptosis and pyroptosis in glioblastoma multiforme cells

doi: 10.1080/14756366.2025.2594158

Figure Lengend Snippet: Results of flow cytometry analysis of caspase 8 activation in A172 (A) and U-87 MG (B) glioblastoma cells after 24 h of incubation with compounds. Graphs visualise the cytograms from the compound analysis A172 (C) and U-87 MG (D). Means ± SD are shown with N = 4. ANOVA tests were used to demonstrate differences between cells treated with compounds and control. ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001.

Article Snippet: Human glioblastoma cell lines (A172 CRL-1620 TM , U-87 MG HTB-14 TM , U-118 MG HTB-15 TM , T98G CRL-1690 TM ) and the mouse astrocyte cell line (C8-D1A CRL-2541 TM ) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Flow Cytometry, Activation Assay, Incubation, Control

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Sulphonamide derivatives with a triazine core as novel inducers of apoptosis and pyroptosis in glioblastoma multiforme cells

doi: 10.1080/14756366.2025.2594158

Figure Lengend Snippet: Results of flow cytometry analysis of caspase 9 activation in A172 (A) and U-87 MG (B) glioblastoma cells after 24 h of incubation with compounds. Graphs visualise the cytograms from the compound analysis A172 (C) and U-87 MG (D). Means ± SD are shown with N = 4. ANOVA tests were used to demonstrate differences between cells treated with compounds and control. *** P ≤ 0.001; **** P ≤ 0.0001.

Article Snippet: Human glioblastoma cell lines (A172 CRL-1620 TM , U-87 MG HTB-14 TM , U-118 MG HTB-15 TM , T98G CRL-1690 TM ) and the mouse astrocyte cell line (C8-D1A CRL-2541 TM ) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Flow Cytometry, Activation Assay, Incubation, Control

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Sulphonamide derivatives with a triazine core as novel inducers of apoptosis and pyroptosis in glioblastoma multiforme cells

doi: 10.1080/14756366.2025.2594158

Figure Lengend Snippet: Results of flow cytometry analysis of caspase 3/7 activation in A172 (A) and U-87 MG (B) glioblastoma cells after 24 h of incubation with compounds. Graphs visualise the cytograms from the compound analysis A172 (C) and U-87 MG (D). Means ± SD are shown with N = 4. ANOVA tests were used to demonstrate differences between cells treated with compounds and control. * P ≤ 0.05; *** P ≤ 0.001; **** P ≤ 0.0001.

Article Snippet: Human glioblastoma cell lines (A172 CRL-1620 TM , U-87 MG HTB-14 TM , U-118 MG HTB-15 TM , T98G CRL-1690 TM ) and the mouse astrocyte cell line (C8-D1A CRL-2541 TM ) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Flow Cytometry, Activation Assay, Incubation, Control

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Sulphonamide derivatives with a triazine core as novel inducers of apoptosis and pyroptosis in glioblastoma multiforme cells

doi: 10.1080/14756366.2025.2594158

Figure Lengend Snippet: Results of H2DCFDA and DAPI double staining of glioblastoma cell lines after 30 min of compound treatment. Representative fluorescence microscope images are shown (A) - A172, and (C) U-87 MG cell lines. The graphs show the minimum, maximum and average fluorescence of each sample tested (B) - A172, and (D) U-87 MG. ANOVA tests were used to show differences between control and compound-treated cells. N > 100; ** P ≤ 0.01; **** P ≤ 0.0001.

Article Snippet: Human glioblastoma cell lines (A172 CRL-1620 TM , U-87 MG HTB-14 TM , U-118 MG HTB-15 TM , T98G CRL-1690 TM ) and the mouse astrocyte cell line (C8-D1A CRL-2541 TM ) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Double Staining, Fluorescence, Microscopy, Control

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Sulphonamide derivatives with a triazine core as novel inducers of apoptosis and pyroptosis in glioblastoma multiforme cells

doi: 10.1080/14756366.2025.2594158

Figure Lengend Snippet: Results of flow cytometry analysis of caspase 1 activation of A172 ( A ) and U-87 MG ( B ) glioblastoma cells after 24 h of incubation with compounds. Graphs visualise the cytograms from the compound analysis A172 ( C ) and U-87 MG ( D ). Means ± SD are shown with N = 4. ANOVA tests were used to demonstrate differences between cells treated with compounds and control. **** P ≤ 0.0001 ( E ) Confocal microscope images with colocalization of NF-κB (red) with the nucleus (DAPI, blue) under treatment of TMZ, MM118 and MM119 or not (control). In vivo evaluation of compounds MM118 and MM119. The confocal images were acquired using a 63x oil immersion objective, N = 3.

Article Snippet: Human glioblastoma cell lines (A172 CRL-1620 TM , U-87 MG HTB-14 TM , U-118 MG HTB-15 TM , T98G CRL-1690 TM ) and the mouse astrocyte cell line (C8-D1A CRL-2541 TM ) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Flow Cytometry, Activation Assay, Incubation, Control, Microscopy, In Vivo

Journal: Blood Advances

Article Title: Metabolically aberrant extracellular mitochondria induce oxidative endotheliopathy in traumatic brain injury ∗

doi: 10.1182/bloodadvances.2024015296

Figure Lengend Snippet: ExMt caused endothelial injury in noninjured mice. (A) Cerebral vascular permeability detected by the Evans blue extravasation, (B) tissue edema detected by water contents, and (C) plasma levels of VWF (VWF:Ag) measured at 30 minutes after mice were infused with 1.6 × 10 7 live or fixed exMt per mouse (n = 6-12 per group, 1-way ANOVA). VWF:Ag in exMt-infused mice was presented as the percentage increase over that of control mice (set as 100%). These data are therefore presented as bar graphs without individual data dots. (D) Representative images from hematoxylin and eosin (H&E)–stained sections of brains from exMt-infused and control mice; (i) the area that was sectioned (arrowhead, perivascular microbleeds; arrow, cerebral edema; scale bar, 20 μm). (E) Endothelial permeability of the lungs and liver from mice infused with live or fixed exMt measured by Evans blue extravasation (n = 6-12 per group, 1-way ANOVA). (F) Representative H&E-stained sections of the lungs show enlarged perivascular space with accumulated red blood cells from an exMt-infused mouse but not from a control mouse (arrow, perivascular bleeds; ∗vascular lumen; scale bar, 20 μm). (G) BAL proteins measured 30 minutes after mice underwent infusion with exMt or an equal volume of the vehicle control (n = 6 per group, t test). (H) Cerebral vascular permeability of mice infused with exMt from mouse brains and those from T98 cells (n = 6 per group, 1-way ANOVA). Ag, antigen; BAL, bronchoalveolar lavage; f-exMt, exMt fixed by 2% paraformaldehyde; VWF, von Willebrand factor.

Article Snippet: We tested exMt from (1) the human glioblastoma multiforme T98 cells (American Type Culture Collection, Manassas, VA), which served as the source of human glial cells to ensure species matching when human ECs were examined; (2) brains of C57BL/6J-Tg(CAG-Cox8/EGFP)49Rin mice that express green fluorescent protein (GFP) specifically in the mitochondria (mtGFP mice, RIKEN BioResource Center, Ibaraki, Japan) for exMt-EC interaction and endocytosis; and (3) brains from C57BL/6J mice for exMt-induced endotheliopathy.

Techniques: Permeability, Clinical Proteomics, Control, Staining

Journal: Blood Advances

Article Title: Metabolically aberrant extracellular mitochondria induce oxidative endotheliopathy in traumatic brain injury ∗

doi: 10.1182/bloodadvances.2024015296

Figure Lengend Snippet: Metabolic activity of exMt and exMt-endocytosed ECs. (A) T98 cells shed exMt when they were cultured under the serum-starvation condition in a time-dependent manner and some of them bound annexin V or expressed the glial cell marker GFAP (n = 6 per time point, 1-way ANOVA; ∗ P < .01 and ∗∗ P < .05 among groups; # P < .05 in longitudinal comparison). (B) Increasing amounts of mtDNA in the conditioned medium over time from serum-starved ECs in culture (n = 6 per time point, 1-way ANOVA). (C) The dose-dependent ATP production of exMt (n = 6 per group, 1-way ANOVA). (D) ROS production from exMt detected by CellROX Red (n = 6 per group, 1-way ANOVA). ECs were incubated with exMt for 3 hours at 37°C followed by trypsin treatment to remove surface-bound exMt. They were then analyzed for oxygen consumption (E), ROS production (F), ATP production (G), and the JC-1 ratio (H; n = 6-12 per group, 1-way ANOVA). For panels E-H, 0 (control): cells treated with the supernatant from exMt isolation. The case-to-control ratio was 2:1. AA, antimycin; GFAP, glial fibrillary acidic protein; GL, glucose; GO, glucose oxidase; L-Cys, L-cysteine; MFI, mean fluorescence intensity; NAC, N-acetyl cysteine; TBHP, tert-butyl hydroperoxide.

Article Snippet: We tested exMt from (1) the human glioblastoma multiforme T98 cells (American Type Culture Collection, Manassas, VA), which served as the source of human glial cells to ensure species matching when human ECs were examined; (2) brains of C57BL/6J-Tg(CAG-Cox8/EGFP)49Rin mice that express green fluorescent protein (GFP) specifically in the mitochondria (mtGFP mice, RIKEN BioResource Center, Ibaraki, Japan) for exMt-EC interaction and endocytosis; and (3) brains from C57BL/6J mice for exMt-induced endotheliopathy.

Techniques: Activity Assay, Cell Culture, Marker, Comparison, Incubation, Control, Isolation, Fluorescence

Journal: Blood Advances

Article Title: Metabolically aberrant extracellular mitochondria induce oxidative endotheliopathy in traumatic brain injury ∗

doi: 10.1182/bloodadvances.2024015296

Figure Lengend Snippet: ExMt induced coagulation. Levels of CD31 + eEVs (A) and VWF (B) detected in the supernatant of cultured human ECs treated with T98-exMt for 3 hours at 37°C (n = 6-12 per group, 1-way ANOVA). (C) Annexin V–bound eEVs in the conditioned medium of ECs stimulated with live exMTs in the absence and presence of 50 nM of CL (n = 6-12 per group, 1-way ANOVA). The EV-rich pellets and the supernatant of the conditioned medium from exMt-stimulated ECs were analyzed for (D) thrombin generation (n = 6-12 per group, t test) and (E) clotting time (n = 6-12 per group, 1-way ANOVA). C57BL/6J mice were infused with 1.6 × 10 7 exMt per mouse. Blood samples were collected 30 minutes after infusion and analyzed for activated FXa on CD31 + eEVs (F), clotting time (G), and levels of D-dimer (H). Control mice received either the equal volume of supernatant or exMt (n = 6-12 per group, 1-way ANOVA). (I) Annexin V binding to live and fixed exMt, which was blocked by 50 nM of CL (n = 6-12 per group, 1-way ANOVA). For panels A-I, the case-to-control ratio was 2:1.

Article Snippet: We tested exMt from (1) the human glioblastoma multiforme T98 cells (American Type Culture Collection, Manassas, VA), which served as the source of human glial cells to ensure species matching when human ECs were examined; (2) brains of C57BL/6J-Tg(CAG-Cox8/EGFP)49Rin mice that express green fluorescent protein (GFP) specifically in the mitochondria (mtGFP mice, RIKEN BioResource Center, Ibaraki, Japan) for exMt-EC interaction and endocytosis; and (3) brains from C57BL/6J mice for exMt-induced endotheliopathy.

Techniques: Coagulation, Cell Culture, Control, Binding Assay

Journal: Biomolecules

Article Title: ADAR2 Protein Is Associated with Overall Survival in GBM Patients and Its Decrease Triggers the Anchorage-Independent Cell Growth Signature

doi: 10.3390/biom12081142

Figure Lengend Snippet: ADAR2 correlates with patients’ overall survival. ( a ) IHC analysis (H&E and the corresponding ADAR2 antibody staining) of two glioblastoma tissues (examples of high and low ADAR2 levels). Subpanels A and B show a representative GBM case with high ADAR2 expression; in subpanels C and D, a GBM case with low ADAR2 expression is shown; scale bar: 200 µm. ( b ) Kaplan–Meier curve comparing the survival of GBM patients (n = 39) stratified by ADAR2 levels. The red and blue lines represent low and high ADAR2 expression, respectively, following the scores indicated in Material and Methods ( p = 0.0005; HR 3.954; 95% CI from 1834 to 8525).

Article Snippet: We analyzed the endogenous ADAR2 level (mRNA and protein) in several human GBM cell lines (A172, T98G, U118MG, U87MG, LN18, and U138-MG) that show diverse abilities to give rise to a growing tumor mass if implanted in a mouse model ( www.atcc.org , accessed on 1 January 2022) ( a,b).

Techniques: Staining, Expressing

Journal: Biomolecules

Article Title: ADAR2 Protein Is Associated with Overall Survival in GBM Patients and Its Decrease Triggers the Anchorage-Independent Cell Growth Signature

doi: 10.3390/biom12081142

Figure Lengend Snippet: ADAR2 expression level and activity across GBM cell lines. ( a ) ADAR2 expression (qRT-PCR) in normal brain (fetal and adult), astrocytes, and glioblastoma cell lines (T98G, U138-MG, U87-MG, U118-MG, LN-18, and A172). Ct values were normalized to GAPDH mRNA levels. Mean ± standard deviation ( n = 3), values are representative as means ± SD, * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001. Of note, ADAR2 activity is more robust in the whole brain (either adult or fetal) because of neuronal cells, where ADAR2 is highly active. ( b ) Western blotting analysis showing the ADAR2 level in glioblastoma cell lines. No control was added as the ADAR2 level was too high. ( c ) The recoding editing index (REI) in normal astrocytes (×10), T98-G, U87-MG, U118-MG, LN-18, and A172 glioblastoma cells lines is shown. REI values were calculated as the weighted average of editing levels over all known recording sites from the highly accurate list.

Article Snippet: We analyzed the endogenous ADAR2 level (mRNA and protein) in several human GBM cell lines (A172, T98G, U118MG, U87MG, LN18, and U138-MG) that show diverse abilities to give rise to a growing tumor mass if implanted in a mouse model ( www.atcc.org , accessed on 1 January 2022) ( a,b).

Techniques: Expressing, Activity Assay, Quantitative RT-PCR, Standard Deviation, Western Blot, Control

Journal: Cells

Article Title: PDCD10 Is a Key Player in TMZ-Resistance and Tumor Cell Regrowth: Insights into Its Underlying Mechanism in Glioblastoma Cells

doi: 10.3390/cells13171442

Figure Lengend Snippet: Knockdown of PDCD10 confers TMZ-resistance on GBM cells. ( A ) Confirmation of PDCD10 knockdown in lentiviral transduced U87 and T98g cells by RT 2 -PCR ( a ), western blot ( b ), and semi-quantitation of the blots ( c ). ev and sh: empty vector- and PDCD10 shRNA-transduced cells, respectively. IOD: integrated optical density. *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with ev. ( B ) Knockdown of PDCD10 in GBM cells leads to a resistance to TMZ-induced cell death. U87 and T98g cells received the treatment with 150 µM ( a ) and 300 µM ( b ) of TMZ, respectively, for 72 h. Thereafter, TMZ was washed-out. Remaining viable cells were cultured in the TMZ-free medium for 3 d, which is defined as the post-treatment phase. Control cells (C) were treated with vehicle DMSO (0.1% and 0.2% for U87 and T98g, respectively). MTT assay was performed to determine the viability of cells at 72 h after TMZ treatment (treatment phase) and 3 d after washing-out of TMZ (post-treatment phase). *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with ev; ##, p < 0.01; ###, p < 0.0001, compared with evC in the same phase; +++, p < 0.001, compared with corresponding group in treatment phase.

Article Snippet: Empty vector transduced cells in U87 (evU87) (Thermo Scientific, Waltham, MA, USA, cat# RHS4750) and T98g (evT98g) (OriGene, Rockville, MD, USA, cat# TR30021) served as controls.

Techniques: Knockdown, Western Blot, Quantitation Assay, Plasmid Preparation, shRNA, Cell Culture, Control, MTT Assay

Journal: Cells

Article Title: PDCD10 Is a Key Player in TMZ-Resistance and Tumor Cell Regrowth: Insights into Its Underlying Mechanism in Glioblastoma Cells

doi: 10.3390/cells13171442

Figure Lengend Snippet: Knockdown of PDCD10 enhances cell viability after rechallenge with TMZ in regrown cells (RG) generated from the established acquired TMZ-resistant model. ( A ) Confirmation of PDCD10 knockdown in shU87-RG and shT98g-RG cells by RT 2 -PCR ( a ) and by FACS of respective transduced cells that expressed red-fluorescence protein (RFP) and green-fluorescence protein (GFP) ( b ). *, p < 0.05, compared with ev. ( B ) MTT assay in RG cells in treatment phase and post-treatment phase. MTT assay was performed with ev/shU87 and ev/shT98g cells that received the treatment with 150 µM ( a ) and 300 µM ( b ) of TMZ, respectively, for 72 h (treatment phase) and 2 d and 4 d after washing-out TMZ (post-treatment phase, without reseeding). Control cells (C) received vehicle DMSO (0.1% and 0.2% for U87 and T98g, respectively). *, p < 0.05; ***, p < 0.001, compared with evRG C (72 h); ###, p < 0.001, compared with evRG-TMZ (72 h). ( C ) MTT assay in RG cells in a second post-treatment model with reseeding. ev/shU87-RG and ev/shT98g-RG cells received the treatment with 150 µM ( a ) and 300 µM ( b ) of TMZ, respectively, for 72 h. Thereafter, TMZ–containing media and dead cells were washed-out, and the viable cells were harvested and reseeded at the same density, followed by 2, 4, and 6 d of culture in drug-free medium. A significantly more rapid regrowth was observed in both TMZ-treated shU87-RG and shT98g-RG cells, compared with the corresponding evRG cells after reseeding and culturing in drug-free media. *, p < 0.05; ***, p < 0.001, compared with corresponding evRG.

Article Snippet: Empty vector transduced cells in U87 (evU87) (Thermo Scientific, Waltham, MA, USA, cat# RHS4750) and T98g (evT98g) (OriGene, Rockville, MD, USA, cat# TR30021) served as controls.

Techniques: Knockdown, Generated, Fluorescence, MTT Assay, Control

Journal: Cells

Article Title: PDCD10 Is a Key Player in TMZ-Resistance and Tumor Cell Regrowth: Insights into Its Underlying Mechanism in Glioblastoma Cells

doi: 10.3390/cells13171442

Figure Lengend Snippet: Overexpression of PDCD10 sensitizes GBM cells to TMZ treatment. ( A ) Confirmation of overexpression of PDCD10 in lentiviral transduced U87 and T98g cells by RT 2 -PCR ( a ) and western blot ( b ) and semi-quantitation of the blots ( c ). Western blotting with anti-V5 antibody distinguishes between the expression of transgenic C-terminal V5-tagged PDCD10 protein and endogenous protein. ev and ox: empty vector-transduced and PDCD10-overexpressing cells, respectively. IOD: integrated optical density. *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with ev. ( B ) Overexpression of PDCD10 significantly reduces cell viability in a concentration-dependent manner after 72 h of TMZ treatment in both oxU87 ( a ) and oxT98g ( b ) cells. Control cells (C) were treated with vehicle DMSO (0.1% and 0.2% for U87 and T98g, respectively). *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with corresponding ev groups. ( C ) Overexpression of PDCD10 sensitizes GBM cells to TMZ treatment 72 h after TMZ treatment (treatment phase) and 3 d after washing-out TMZ (post-treatment phase). ev/oxU87 and ev/oxT98g cells received the treatment with 150 µM ( a ) and 300 µM ( b ) of TMZ for 72 h, respectively. Thereafter, TMZ-containing medium and dead cells were washed-out and the viable cells were further cultured in drug-free medium for 3 d followed by MTT assay. Control cells (C) were treated with vehicle DMSO (0.1% and 0.2% for U87 and T98g, respectively). *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with corresponding ev groups; +++, p < 0.001, compared with corresponding evC in the treatment phase; #, p < 0.05, ###, p < 0.001, compared with corresponding evC in the same phase.

Article Snippet: Empty vector transduced cells in U87 (evU87) (Thermo Scientific, Waltham, MA, USA, cat# RHS4750) and T98g (evT98g) (OriGene, Rockville, MD, USA, cat# TR30021) served as controls.

Techniques: Over Expression, Western Blot, Quantitation Assay, Expressing, Transgenic Assay, Plasmid Preparation, Concentration Assay, Control, Cell Culture, MTT Assay

Journal: Cells

Article Title: PDCD10 Is a Key Player in TMZ-Resistance and Tumor Cell Regrowth: Insights into Its Underlying Mechanism in Glioblastoma Cells

doi: 10.3390/cells13171442

Figure Lengend Snippet: DNA replication in response to TMZ treatment is dependent on PDCD10 expression. DNA replication was detected by EdU incorporation followed by FACS at 72 h of TMZ treatment (treatment phase) and at 3 d after TMZ-washing-out and -culturing in drug-washout media (post-treatment phase). ev/shT98g-RG and ev/oxT98g cells received 500 and 300 µM TMZ, respectively. ( A , C ) Histograms of EdU-positive (EdU+) and -negative (EdU−) cell populations in ev/shT98g-RG and ev/oxT98g cells, respectively. ( B , D ) Bar graphs of EdU+/− populations based on the corresponding histograms in ( A , C ). Knockdown of PDCD10 leads to an increase in DNA replication in both the treatment and post-treatment phases of T98g-RG cells, whereas overexpression of PDCD10 suppresses DNA replication in response to TMZ treatment. The data are representative of at least three independent experiments.

Article Snippet: Empty vector transduced cells in U87 (evU87) (Thermo Scientific, Waltham, MA, USA, cat# RHS4750) and T98g (evT98g) (OriGene, Rockville, MD, USA, cat# TR30021) served as controls.

Techniques: Expressing, Knockdown, Over Expression

Journal: Cells

Article Title: PDCD10 Is a Key Player in TMZ-Resistance and Tumor Cell Regrowth: Insights into Its Underlying Mechanism in Glioblastoma Cells

doi: 10.3390/cells13171442

Figure Lengend Snippet: Knockdown of PDCD10 in T98g-RG cells leads to deregulation of DNA damage response (DDR) genes. ev/shT98g-RG and ev/oxT98g cells received 300 µM of TMZ or vehicle (0.2% DMSO) treatment (no TMZ treatment). Cells were harvested for PCR detection of DDR genes after 72 h of TMZ treatment (treatment phase) and at 3 d after TMZ-washing-out and culturing in drug-free media (post-treatment phase). ( A ) Expression of MGMT in T98g-RG ( a ) cells and ev/oxT98g ( b ) cells in the no treatment, treatment (300 µM, 72 h), and post-treatment (3 d after washing out) phases. ( B ) Western blot ( a ) and semi-quantitation of the blots ( b ) of the MGMT protein expression in ev/shT98g-RG and ev/oxT98g cells. ( C ) Expression of DDR genes ( MSH2 , MSH6, and PMS2 ) in T98g-RG cells in the no treatment ( a ), treatment ( b ), and post-treatment phases ( c ), respectively. ( D ) Expression of DDR genes in ev/oxT98g cells in the no treatment ( a ), treatment ( b ), and post-treatment phases ( c ), respectively. *, p < 0.05; **, p < 0.01 and ***, p < 0.001, compared with corresponding ev.

Article Snippet: Empty vector transduced cells in U87 (evU87) (Thermo Scientific, Waltham, MA, USA, cat# RHS4750) and T98g (evT98g) (OriGene, Rockville, MD, USA, cat# TR30021) served as controls.

Techniques: Knockdown, Expressing, Western Blot, Quantitation Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Structural mechanism of Myb–MuvB assembly

doi: 10.1073/pnas.1808136115

Figure Lengend Snippet: The B-Myb C-terminal domain is necessary and sufficient for MuvB association. (A) Domain architecture of Myb proteins (sequence numbering for human B-Myb), including a DNA-binding domain (DBD), transactivation domain (TAD), and a negative regulatory domain (NRD). The MuvB binding domain (MBD, residues 657–688) investigated here is within the NRD and has the aligned sequence. The secondary structure and amino acids that interact with MuvB (asterisks) are determined from the crystal structure in this study. Amino acids that are highly conserved in vertebrate B-Myb orthologs and Drosophila Myb (dMyb) are highlighted green, with changes at these positions in A-Myb and c-Myb shown in red and blue (SI Appendix, Fig. S1). (B) T98G cells were transfected with plasmids encoding the indicated GFP-B-Myb fusion protein. Vector expresses GFP only. Lysates were immunoprecipitated with an anti-LIN37 antibody, and Western blots performed to assay the protein of interest. The asterisk marks Ig bands from the primary antibody.

Article Snippet: Human T98G cells (#CRL 1690; ATCC) were transiently transfected with pcDNA3.1 vectors encoding a GFP-only control, or GFP-tagged wild-type or mutant B-Myb proteins.

Techniques: Sequencing, Binding Assay, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: PLoS ONE

Article Title: Characterization of a Novel Anti-Cancer Compound for Astrocytomas

doi: 10.1371/journal.pone.0108166

Figure Lengend Snippet: (A) The structure of CC-I. (B) Cytotoxicity of CC-I in in vitro . Human astrocytoma cell lines were cultured with different doses of CC-I for 3 days and then the cytotoxicity was determined by SRB assay. The LC 50 of CC-I to SW1088 cell lines (13.6 µM) are significantly different with the LC 50 of CC-I to U87-MG and CCF-STTG1 cell lines (23.6 µM and 25.4 µM) (p<0.001).

Article Snippet: Human astrocytoma cells (SW1088-grade III, U87-MG-grade IV, CCF-STTG1-grade IV, T98G-grade IV, LN-18-grade IV) were ordered from American Type Culture Collection (ATCC, Manassas, VA) and maintained in DMEM (Gibco by Life Technologies, catalog 11885) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 0.29 mg/mL L-glutamine, and 10% FBS.

Techniques: In Vitro, Cell Culture, Sulforhodamine B Assay

Journal: PLoS ONE

Article Title: Characterization of a Novel Anti-Cancer Compound for Astrocytomas

doi: 10.1371/journal.pone.0108166

Figure Lengend Snippet: (A) Mice were implanted with ten million cells with the SW1088 or CCF-STTG1 cells. The starting tumor size for the CCF-STTG1 cells ranged from 80–100 mm 3 . The SW1088 cells grew more slowly so CC-I treatment was started when the tumors reached 30 mm 3 . CC-I was injected intraperitoneally at a concentration of 25 mg/kg body weight once a week for 7 weeks (n = 7∼10). The control group was given PBS in the same volume and regimen (n = 3–8). The tumor slowly reoccurred in the TMZ-sensitive SW1088 astrocytoma injected nude mice but did not reoccur in the TMZ resistant CCF-STTG1 injected nude mice when CC-I was discontinued (beyond 7 weeks). CC-I inhibited the tumor growth and was not lethal in any of the treatment groups. Some error bars are too small to be visible. (B) Mean body weight of mice is presented in grams. Some error bars are too small to be visible.

Article Snippet: Human astrocytoma cells (SW1088-grade III, U87-MG-grade IV, CCF-STTG1-grade IV, T98G-grade IV, LN-18-grade IV) were ordered from American Type Culture Collection (ATCC, Manassas, VA) and maintained in DMEM (Gibco by Life Technologies, catalog 11885) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 0.29 mg/mL L-glutamine, and 10% FBS.

Techniques: Injection, Concentration Assay, Control

Journal: PLoS ONE

Article Title: Characterization of a Novel Anti-Cancer Compound for Astrocytomas

doi: 10.1371/journal.pone.0108166

Figure Lengend Snippet: (A) Representative MRI images taken with T1-weighted MRI contrast (7T MR imaging system) after intracranial tumor formation (one-three weeks post-implantation of astrocytoma cells) or after tumor formation followed by injection of CC-I (25 mg/kg body weight) for 7 weeks. CC-I completely inhibited tumor growth in both astrocytoma cell lines. (B) Kaplan-Meier survival graph of intracranial brain tumor mice after the administration of CC-I. CC-I extends the survival of the mice when compared to the untreated mice (n = 9 or 11) (p<0.0001). None of the mice which received PBS (control) survived after 30 days and median survival of all those animals was 20 days (n = 3 or 4). (C) Liver and kidney toxicity of CC-I. The liver and kidney toxicity (total bilirubin, blood urea nitrogen (BUN), creatine, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase) were determined using an automated chemistry analyzer machine (Roche Cobase MIRA) and kits manufactured by Thermo Electron. These data indicate no liver or kidney toxicity by CC-I in nude mice. Toxicity data displayed as means ± SEM. (D) Mean body weight of mice in grams.

Article Snippet: Human astrocytoma cells (SW1088-grade III, U87-MG-grade IV, CCF-STTG1-grade IV, T98G-grade IV, LN-18-grade IV) were ordered from American Type Culture Collection (ATCC, Manassas, VA) and maintained in DMEM (Gibco by Life Technologies, catalog 11885) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 0.29 mg/mL L-glutamine, and 10% FBS.

Techniques: Imaging, Injection, Control

Journal: PLoS ONE

Article Title: Characterization of a Novel Anti-Cancer Compound for Astrocytomas

doi: 10.1371/journal.pone.0108166

Figure Lengend Snippet: (A) TMZ-resistant human CCF-STTG1 and T98G cell lines were cultured for 3 days with CC-I and other similar structure topoisomerase II inhibitor (merbarone) followed by cytotoxicity measurement by SRB assay. CC-I showed greater toxicity than merbarone on the astrocytomas. The symbols indicate a significant difference between the merbarone treated and CC-I treated groups (**p<0.01; ***p<0.001). (B) The MGMT methylated (T98G, CCF-STTG1) or un-methylated (LN-18) astrocytoma cell lines were cultured for 3 days with CC-I and determined cytotoxicity by SRB assay. T98G cells have methylated MGMT promoter, but show weak MGMT expression. CC-I is more cytotoxic to LN-18 cells which has un-methylated MGMT promoter and MGMT expression. The symbol (***) indicates the most difference between the cells (p<0.001).

Article Snippet: Human astrocytoma cells (SW1088-grade III, U87-MG-grade IV, CCF-STTG1-grade IV, T98G-grade IV, LN-18-grade IV) were ordered from American Type Culture Collection (ATCC, Manassas, VA) and maintained in DMEM (Gibco by Life Technologies, catalog 11885) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 0.29 mg/mL L-glutamine, and 10% FBS.

Techniques: Cell Culture, Sulforhodamine B Assay, Methylation, Expressing