human glioblastoma astrocytoma cells u 373 mg  (ATCC)

Journal: Nutrients

Article Title: Supercritical CO 2 -Derived Tomato Extract Activates Signaling Pathways to Reduce Oxidative Stress and Inflammation in Astrocyte Cells

doi: 10.3390/nu18091464

Figure Lengend Snippet: Effect of supercritical CO 2 tomato extract (sCO 2 TE) on cell viability. Human glioblastoma astrocyte cells U-373 were treated with sCO 2 TE (100 μg/mL), conventional tomato extract (CTE, 100 μg/mL) or sodium arsenite (AsNaO 2 , 200 μM). The number of viable cells was determined by Trypan Blue (TB) exclusion test. Data are reported as the mean ± standard deviation (SD) among 10 independent experiments ( A ). Cell proliferation was measured by 3-(4,5-dimethylthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data are reported as the mean ± standard deviation (SD) among 5 independent experiments performed in triplicate and represent cell viability as a percentage of vehicle ( B ).

Article Snippet: Human glioblastoma astrocytoma cells U-373 MG (American Type Culture Collection, ATCC, Manassas, VA, USA) were grown in DMEM F-12 medium (Sigma-Aldrich, Milan, Italy) supplemented with 10% fetal bovine serum (FBS, Aurogene S.r.l., Rome, Italy), 100 units/mL penicillin and 10 mg/mL streptomycin (Aurogene S.r.l.) and maintained in a humified atmosphere of 37 °C and 5% CO 2 .

Techniques: Standard Deviation, MTT Assay

Journal: Nutrients

Article Title: Supercritical CO 2 -Derived Tomato Extract Activates Signaling Pathways to Reduce Oxidative Stress and Inflammation in Astrocyte Cells

doi: 10.3390/nu18091464

Figure Lengend Snippet: Effect of supercritical CO 2 tomato extract (sCO 2 TE) on DNA. Human glioblastoma astrocyte cells U-373 were treated with sCO 2 TE (100 μg/mL), conventional tomato extract (CTE, 100 μg/mL) or sodium arsenite (AsNaO 2 , 200 μM) and, after staining with propidium iodide (PI), were analyzed by flow cytometric analysis to evaluate cell cycle phases. Data are reported as the mean ± standard deviation (SD) among 3 independent experiments.

Article Snippet: Human glioblastoma astrocytoma cells U-373 MG (American Type Culture Collection, ATCC, Manassas, VA, USA) were grown in DMEM F-12 medium (Sigma-Aldrich, Milan, Italy) supplemented with 10% fetal bovine serum (FBS, Aurogene S.r.l., Rome, Italy), 100 units/mL penicillin and 10 mg/mL streptomycin (Aurogene S.r.l.) and maintained in a humified atmosphere of 37 °C and 5% CO 2 .

Techniques: Staining, Standard Deviation

Journal: Nutrients

Article Title: Supercritical CO 2 -Derived Tomato Extract Activates Signaling Pathways to Reduce Oxidative Stress and Inflammation in Astrocyte Cells

doi: 10.3390/nu18091464

Figure Lengend Snippet: Analysis of intracellular reactive oxygen species (ROS) after supercritical CO 2 tomato extract (sCO 2 TE) treatment. Human glioblastoma astrocyte cells U-373 were treated with sCO 2 TE (100 μg/mL), conventional tomato extract (CTE, 100 μg/mL) or sodium arsenite (AsNaO 2 , 200 μM), and alternatively they were pre-treated with sCO 2 TE (100 μg/mL) and then stimulated with AsNaO 2 (200 μM). After treatments, ROS production was assessed using the DCFH-DA fluorescent probe. Flow cytometric histograms show measurement of ROS levels through flow cytometry analysis ( A ). Quantified ROS levels: histograms show the mean fluorescence intensity (MFI) ( B ). The results are presented as mean ± SD from 3 independent experiments. Statistical analysis indicates: * p < 0.05 and *** p < 0.001.

Article Snippet: Human glioblastoma astrocytoma cells U-373 MG (American Type Culture Collection, ATCC, Manassas, VA, USA) were grown in DMEM F-12 medium (Sigma-Aldrich, Milan, Italy) supplemented with 10% fetal bovine serum (FBS, Aurogene S.r.l., Rome, Italy), 100 units/mL penicillin and 10 mg/mL streptomycin (Aurogene S.r.l.) and maintained in a humified atmosphere of 37 °C and 5% CO 2 .

Techniques: Flow Cytometry, Fluorescence

Journal: Nutrients

Article Title: Supercritical CO 2 -Derived Tomato Extract Activates Signaling Pathways to Reduce Oxidative Stress and Inflammation in Astrocyte Cells

doi: 10.3390/nu18091464

Figure Lengend Snippet: Analysis of lipid peroxidation after supercritical CO 2 tomato extract (sCO 2 TE) treatment. Human glioblastoma astrocyte cells U-373 were treated with sCO 2 TE (100 μg/mL), conventional tomato extract (CTE, 100 μg/mL) or sodium arsenite (AsNaO 2 , 200 μM), and alternatively they were pre-treated with sCO 2 TE (100 μg/mL) and then stimulated with AsNaO 2 (200 μM). After treatments, lipid peroxidation was evaluated using BODIPY 581/591 C11probe (Lipid Peroxidation Sensor that results in a FITC emission). Flow cytometric histograms show measurement of lipid peroxidation through flow cytometry analysis ( A ). Quantified lipid peroxidation, histograms show the mean fluorescence intensity (MFI) ( B ). The results are presented as mean ± SD from 3 independent experiments. Statistical analysis indicates: **** p < 0.0001.

Article Snippet: Human glioblastoma astrocytoma cells U-373 MG (American Type Culture Collection, ATCC, Manassas, VA, USA) were grown in DMEM F-12 medium (Sigma-Aldrich, Milan, Italy) supplemented with 10% fetal bovine serum (FBS, Aurogene S.r.l., Rome, Italy), 100 units/mL penicillin and 10 mg/mL streptomycin (Aurogene S.r.l.) and maintained in a humified atmosphere of 37 °C and 5% CO 2 .

Techniques: Flow Cytometry, Fluorescence

Journal: Nutrients

Article Title: Supercritical CO 2 -Derived Tomato Extract Activates Signaling Pathways to Reduce Oxidative Stress and Inflammation in Astrocyte Cells

doi: 10.3390/nu18091464

Figure Lengend Snippet: Analysis of NRF-2 phosphorylation after supercritical CO 2 tomato extract (sCO 2 TE) treatment. Human glioblastoma astrocyte cells U-373 were treated with sCO 2 TE (100 μg/mL), conventional tomato extract (CTE, 100 μg/mL) or sodium arsenite (AsNaO 2 , 200 μM), and alternatively they were pre-treated with sCO 2 TE (100 μg/mL) and then stimulated with AsNaO 2 (200 μM). After treatments, cells were lysed and analyzed by Western blot. Nuclear cell extracts were analyzed to verify phosphorylated NRF2 using rabbit anti-phospho-NRF2 Ab. As a control, for loading and purity of preparation, membrane was stripped and reprobed with polyclonal anti-HISTONE H1 Ab. Densitometric phospho-NRF2/HISTONE H1 ratios are shown in the right panel. Results represent the mean ± SD from 3 independent experiments. Statistical analysis indicates: ** p < 0.01; **** p < 0.0001.

Article Snippet: Human glioblastoma astrocytoma cells U-373 MG (American Type Culture Collection, ATCC, Manassas, VA, USA) were grown in DMEM F-12 medium (Sigma-Aldrich, Milan, Italy) supplemented with 10% fetal bovine serum (FBS, Aurogene S.r.l., Rome, Italy), 100 units/mL penicillin and 10 mg/mL streptomycin (Aurogene S.r.l.) and maintained in a humified atmosphere of 37 °C and 5% CO 2 .

Techniques: Phospho-proteomics, Western Blot, Control, Membrane

Journal: Nutrients

Article Title: Supercritical CO 2 -Derived Tomato Extract Activates Signaling Pathways to Reduce Oxidative Stress and Inflammation in Astrocyte Cells

doi: 10.3390/nu18091464

Figure Lengend Snippet: Analysis of MAPK signaling and NF-κB activation after supercritical CO 2 tomato extract (sCO 2 TE) treatment. Human glioblastoma astrocyte cells U-373 were treated with sCO 2 TE (100 μg/mL), conventional tomato extract (CTE, 100 μg/mL) or LPS (100 ng/mL), and alternatively they were pre-treated with sCO 2 TE (100 μg/mL) and then stimulated with LPS (100 ng/mL). After treatments cells were lysed and analyzed by Western blot. ( A ) Whole cell extracts were analyzed to verify phosphorylated-ERK expression using rabbit anti-phospho-ERK1/2 Ab. ( B ) Nuclear cell extracts were analyzed to verify phosphorylated-NF-kB-p65 expression using rabbit anti-phospho-NF-kB-p65 Ab. As a control, for loading and purity of preparation, membrane was stripped and reprobed with polyclonal anti-HISTONE H1 Ab. Densitometric phospho-ERK/total ERK and phospho-NF-kB-p65/HISTONE H1 ratios are shown in the right panels. Results represent the mean ± SD from 3 independent experiments. Statistical analysis indicates: ** p < 0.01; **** p < 0.0001.

Article Snippet: Human glioblastoma astrocytoma cells U-373 MG (American Type Culture Collection, ATCC, Manassas, VA, USA) were grown in DMEM F-12 medium (Sigma-Aldrich, Milan, Italy) supplemented with 10% fetal bovine serum (FBS, Aurogene S.r.l., Rome, Italy), 100 units/mL penicillin and 10 mg/mL streptomycin (Aurogene S.r.l.) and maintained in a humified atmosphere of 37 °C and 5% CO 2 .

Techniques: Activation Assay, Western Blot, Expressing, Control, Membrane

Journal: Redox Biology

Article Title: Alexander disease mutations differentially sensitize glial fibrillary acidic protein (GFAP) to posttranslational modifications and network disruption by oxidants

doi: 10.1016/j.redox.2026.104103

Figure Lengend Snippet: Analysis of disulfide-mediated oligomerization of GFAP AxD mutants under basal and oxidizing conditions in U-87 MG cells . U-87 MG astrocytoma cells expressing GFP-GFAP wt or AxD mutants were lysed, and cell lysates were prepared in the absence (non-reducing conditions) or presence (reducing conditions) of β-mercaptoethanol for western blot analysis. ( A ) Basal oligomerization was examined in cells expressing GFP-GFAP wt or the R239C, R79C, or E373K mutants. The amount of sample loaded was adjusted according to the levels of GFP-GFAP in each lysate as follows: 10 μg total protein for wt and E373K, 2 μg for R79C, and 5 μg for R239C. ( B ) Glutathione levels in U87-MG cells expressing GFP-GFAP wt or AxD mutants. Total (GSH + GSSG) and oxidized glutathione (GSSG) were measured in U87-MG cells expressing GFP-GFAP wt or the AxD mutants, R79C, R239C or E373K. The figure shows normalized data of the three parameters, where values for GFP-GFAP E373K-expressing cells were taken as 100% due to their lower interexperimental variability between independent experiments. Top panel depicts total glutathione results (100% = 3082.4 ± 849.7 pmol/mg protein) and bottom panel shows GSSG concentrations (100% = 115.9 ± 73.3 pmol/mg protein). Statistical analysis was performed with GraphPad Prism v9 using Kruskal-Wallis test and Dunn's post-hoc analysis. ( C ) Oxidant-induced oligomerization was assessed in cells expressing GFP-GFAP wt or R239C treated with 1 mM H 2 O 2 for 30 min, 0.5 mM ONOOH for 15 min, or 50 μM HOCl for 15 min at 37 o C. Sample loading was adjusted according to expression levels: 30 μg total protein for wt and 4 μg for R239C. Asterisks indicate GFP-GFAP wt monomers, and arrowheads indicate oligomers in panels A and C. Molecular mass markers (kDa) are shown on the left of the gels. Dashed lines separate two sections of the same blot. Lower panels in (A) and (C) show long time exposures of the same blot to ensure detection of low-abundance oligomeric bands.

Article Snippet: The U-87 MG glioblastoma astrocytoma cell line was obtained from the American Type Culture Collection (ATCC) and authenticated by short tandem repeat (STR) profiling via microsatellite amplification at Secugen, SL (Madrid, Spain).

Techniques: Expressing, Western Blot

Journal: Redox Biology

Article Title: Alexander disease mutations differentially sensitize glial fibrillary acidic protein (GFAP) to posttranslational modifications and network disruption by oxidants

doi: 10.1016/j.redox.2026.104103

Figure Lengend Snippet: Organization of the GFP-GFAP wt and R239C networks in astrocytoma cells. (A) U-87 MG cells were transfected with GFP-GFAP wt and R239C constructs and treated with the indicated oxidants, as described in the Materials and Methods. After treatment, cells were fixed with 4% (w/v) paraformaldehyde, permeabilized with Triton X-100, blocked with 1% BSA (w/v) and stained with Phalloidin-Alexa Fluor 568 and DAPI. The GFAP networks were directly visualized by the GFP fluorescence. Overall projections of GFP-GFAP (green), Phalloidin (red, actin) and DAPI (blue, nuclei) are shown. Cell contours are outlined in white. Scale bars, 20 μm. (B) Quantification of GFAP-covered area in cells expressing GFP-GFAP wt under control or oxidative treatment conditions. ROIs were defined using phalloidin staining for the cell body and GFP for GFAP-positive filamentous structures. Data represent the mean ± SEM of 20 cells per condition from three independent experiments. (C) Quantitation of effects of the oxidative agents in cells expressing GFP-GFAP R239C. Left graph, percentage of GFP-GFAP R239C-expressing cells containing aggregates under control or treated conditions. At least 150 cells per experiment were analyzed across three independent experiments, each symbol represents an individual experiment. Right graph, average number of aggregates per GFP-GFAP R239C-expressing cell determined in 25 cells per condition across three independent experiments. Aggregates were identified using manual thresholding in ImageJ to include discrete aggregates while excluding bundles. Data are shown as mean ± SEM.

Article Snippet: The U-87 MG glioblastoma astrocytoma cell line was obtained from the American Type Culture Collection (ATCC) and authenticated by short tandem repeat (STR) profiling via microsatellite amplification at Secugen, SL (Madrid, Spain).

Techniques: Transfection, Construct, Staining, Fluorescence, Expressing, Control, Quantitation Assay