u343mg stabilized cell line  (CLS Cell Lines Service GmbH)

Journal: Pharmaceuticals

Article Title: Protopine/Gemcitabine Combination Induces Cytotoxic or Cytoprotective Effects in Cell Type-Specific and Dose-Dependent Manner on Human Cancer and Normal Cells

doi: 10.3390/ph14020090

Figure Lengend Snippet: Effects of protopine (PRO) on cell viability. ( a ) Cell viability of human dermal fibroblasts (HDFs), U343, U87, MIA PaCa-2, PANC-1 and MCF-7 cells was evaluated after treatments for 48 h with different PRO doses, by using MTT. Graph columns represent mean of absorbance values ± standard deviation (SD) measured with MTT assay. In ( b ) Graph columns represent mean of absorbance values ± SD evaluated with crystal violet (CV) assay in the indicated cell lines. Graph columns represent mean ± SD of fluorescence intensity values. Control group contained only PRO vehicle, DMSO. The PRO doses (μM) used in treatments are indicated in x-axis. The symbols * indicate statistical significance versus control group. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The human cancer cell lines that were used in this study were pancreatic carcinoma cells (MIA PaCa-2 and PANC-1; American Type Culture Collection, ATCC), human glioblastoma cells (U343 and U87 MG; CLS Cell Lines Service), breast cancer cells (MCF-7; ATCC) and normal primary dermal fibroblasts HDFs (ATCC, Manassas, VA, USA).

Techniques: Standard Deviation, MTT Assay, Fluorescence

Journal: Cell Death & Disease

Article Title: Histone demethylase KDM4C controls tumorigenesis of glioblastoma by epigenetically regulating p53 and c-Myc

doi: 10.1038/s41419-020-03380-2

Figure Lengend Snippet: A Immunoblot analysis of KDM4C, c-Myc, and its target genes in U87 cells transfected with c-Myc (0.6 μg) without or with the treatment of SD70 (10, 20 μM). B Immunoblot analysis of KDM4C, p53, and its target genes in shCtrl NT and shp53 expressing U87 cells without or with the treatment of SD70 (5 μM).

Article Snippet: U343 (wt p53; 300365) and U251 (mt p53; 300385) cells were purchased from CLS Cell Lines Service GmbH (Eppelheim, Germany).

Techniques: Western Blot, Transfection, Expressing

Journal: Cell Death & Disease

Article Title: Histone demethylase KDM4C controls tumorigenesis of glioblastoma by epigenetically regulating p53 and c-Myc

doi: 10.1038/s41419-020-03380-2

Figure Lengend Snippet: A , B The activity of p21 and Bax luciferase reporter constructs in U87 cells treated with 0.1% DMSO (control) and SD70 following transfection with p53. *** p < 0.001 vs. pcDNA control; $$$ p < 0.001 vs. pcDNA-p53 group, ### p < 0.001 vs. DMSO + pcDNA-p53 group; one-way ANOVA. C The activity of a p21 luciferase reporter construct in U87 cells following transfection with p53 and KDM4C. D – G RT-qPCR showing KDM4s, p53, and p53 target gene mRNA levels in U87 cells treated with shRNA ( D and E ) or SD70 ( F and G ). mRNA expression levels were normalized to GAPDH mRNA levels. H Immunoblotting of KDM4C, p53, and p21 expression in U87 and U251 cells treated with shRNA or SD70 for 24 h. I ChIP-qPCR analysis of p53 and p53K372me1 levels at the PUMA promoter in 0.1% DMSO (control) and SD70 (5 µM) treated U87 cells. Data present the mean ± SD ( n = 3). * p < 0.05 and ** p < 0.01 vs. shCtrl NT or DMSO control; Student’s t- tests.

Article Snippet: U343 (wt p53; 300365) and U251 (mt p53; 300385) cells were purchased from CLS Cell Lines Service GmbH (Eppelheim, Germany).

Techniques: Activity Assay, Luciferase, Construct, Transfection, Quantitative RT-PCR, shRNA, Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: Histone demethylase KDM4C controls tumorigenesis of glioblastoma by epigenetically regulating p53 and c-Myc

doi: 10.1038/s41419-020-03380-2

Figure Lengend Snippet: A , B Immunoblotting of KDM4C, p53, and p53K372me1 in U87 and U251 cells transfected with ( A ) shRNA and ( B ) siRNA. C , D Lysates of cells treated with SD70 (20 μM) ( C ) or HA-KDM4C ( D ) were immunoprecipitated with p53 antibody followed by immunoblotting with antibodies as indicated. Ten percent of total cell lysates used in immunoprecipitation is shown as input. The level of p53K372me1 was quantified relative to p53, and the control levels were set at 1. The asterisk indicates the expected sizes for p53K372me1.

Article Snippet: U343 (wt p53; 300365) and U251 (mt p53; 300385) cells were purchased from CLS Cell Lines Service GmbH (Eppelheim, Germany).

Techniques: Western Blot, Transfection, shRNA, Immunoprecipitation

Journal: Cell Death & Disease

Article Title: Histone demethylase KDM4C controls tumorigenesis of glioblastoma by epigenetically regulating p53 and c-Myc

doi: 10.1038/s41419-020-03380-2

Figure Lengend Snippet: A Immunoblotting of KDM4C, c-Myc, and p53 in U87 TetOn-KDM4C cells expressing shCtrl NT or shKDM4C. B Liquid colony formation assays of U87 TetOn-KDM4C cells expressing shCtrl NT or shKDM4C. * p < 0.05 and ** p < 0.01 vs. Dox (-) group; one-way ANOVA. C – E Xenograft assays of U87 cells with inducible expression of shCtrl NT or shKDM4C ( n = 3). C Representative U87 xenograft tumors isolated from individual animals. D Increase in tumor volume over time. E Final tumor weights of the U87 xenografts. Data present the mean ± S.D. * p < 0.05 and ** p < 0.01 vs. shCtrl NT control; one-way ANOVA. F Representative image of IHC staining of KDM4C and c-Myc in tissues from U87 xenograft model. Scale bar, 100 μm. G Molecular model describing that KDM4C can regulate glioblastoma proliferation and apoptosis by modulating c-Myc and p53.

Article Snippet: U343 (wt p53; 300365) and U251 (mt p53; 300385) cells were purchased from CLS Cell Lines Service GmbH (Eppelheim, Germany).

Techniques: Western Blot, Expressing, Isolation, Immunohistochemistry

Journal: International Journal of Molecular Sciences

Article Title: High Adenosine Extracellular Levels Induce Glioblastoma Aggressive Traits Modulating the Mesenchymal Stromal Cell Secretome

doi: 10.3390/ijms21207706

Figure Lengend Snippet: Effects of ADO on U343MG cell proliferation, expression of stemness genes and cell motility. ( A , B ) U343MG cells were treated with different concentrations of ADO (10 nM to 100 µM) for: 24 h ( A ); or 48 h ( B ). At the end of the treatment, cell proliferation was evaluated, as described in . The data are expressed as percentages relative to untreated cells (CTRL), which were set at 100% (mean ± SEM; N = 3). ( C , D ) The total RNA was extracted from U343MG cells after treatment with ADO (100 nM and 100 µM) for 48 h, and the relative mRNA quantification of the markers SOX2 ( C ) and Oct4 ( D ) was performed by RT-PCR, as described in . The data are expressed as fold changes with respect to basal value set to 1 (mean values ± SEM, N = 2). ( E , F ) U343MG cells were treated with ADO (100 nM or 100 µM) and the healing of the wound was evaluated in the scratch assay. ( E ) Representative images of the scratch wounds at 0 and 24 h. ( F ) The data are expressed as percentage of gap closure after 24 h of treatment compared to the untreated cells (CTRL), set to 100%. The data are represented as the means ± SEM of at least of three independent experiments. The significance of differences was determined by one-way ANOVA, followed by Bonferroni’s post hoc test: * p < 0.05 vs. CTRL.

Article Snippet: Human bone marrow MSCs (BM-MSCs) and glioblastoma stem cells (U343MG) were purchased by CLS (CLS Cell Lines Service GmbH, Eppelheim, Germany).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Wound Healing Assay

Journal: International Journal of Molecular Sciences

Article Title: High Adenosine Extracellular Levels Induce Glioblastoma Aggressive Traits Modulating the Mesenchymal Stromal Cell Secretome

doi: 10.3390/ijms21207706

Figure Lengend Snippet: ADO modulation of GMT process in glioma cells. U343MG cells were treated with ADO (100 nM or 100 µM) for 72 h. ( A , B ) The mRNA expression levels of GMT master genes (Slug, Snail, Twist and ZEB1) ( A ) and the epithelial (CDH1) and mesenchymal (Vimentin and ACTA2) markers ( B ) were determined by Real-Time RT-PCR. The data are expressed as fold changes with respect to basal value set to 1 and are the mean values ± SEM of two independent experiments. ( C , D ) U343MG cells were treated as described above and the protein expression of Epithelial (E-CAD) and Mesenchymal markers (Vimentin and α-SMA) were evaluated by Western blotting. ( C ) One representative blot for each protein is presented and ( D ) the bar graph shows the densitometric analysis of the Western blot performed using ChemiDocTM XRS+ System (BioRad, Hercules, CA, USA). The data are expressed as the fold change vs. the CTRL levels, which were set to 1 and are the mean values ± SEM of three different experiments. The significance was determined by one-way ANOVA, followed by Bonferroni’s post hoc test: * p < 0.05, ** p < 0.01 vs. CTRL.

Article Snippet: Human bone marrow MSCs (BM-MSCs) and glioblastoma stem cells (U343MG) were purchased by CLS (CLS Cell Lines Service GmbH, Eppelheim, Germany).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: High Adenosine Extracellular Levels Induce Glioblastoma Aggressive Traits Modulating the Mesenchymal Stromal Cell Secretome

doi: 10.3390/ijms21207706

Figure Lengend Snippet: Involvement of ERK 1/2 phosphorylation in ADO-mediated induction of GMT traits. ( A ) Time-course analysis of ERK 1/2 phosphorylation in U343MG cells. U343MG cells were treated with ADO (100 nM and 100 µM) for different time (2 min–72 h), and ERK 1/2 phosphorylation was measured by immuno-enzymatic assay. The data are expressed as the percentage versus untreated cells (CTRL) set to 100% ± SEM of at least three independent experiments performed in duplicate. ( B ) Time-course analysis of total ERK 1/2 in U343MG cells. ( C , D ) U343MG cells were treated with ADO (100 nM and 100 µM) in the presence or absence of 1 µM PD184352 for 72 h. mRNA expression levels of GMT master genes (Slug, Snail, Twist and ZEB1) ( C ) and of the Epithelial (CDH1) and Mesenchymal (Vimentin) markers ( D ) were determined by RT-PCR. The data are expressed as fold changes with respect to basal value set to 1 and are the mean values ± SEM of two independent experiments. The significance of the differences was determined by one-way ANOVA, followed by Bonferroni’s post hoc test or two-way ANOVA with Bonferroni correction and two-sided tests for multiple comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. CTRL; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. corresponding ADO treatment.

Article Snippet: Human bone marrow MSCs (BM-MSCs) and glioblastoma stem cells (U343MG) were purchased by CLS (CLS Cell Lines Service GmbH, Eppelheim, Germany).

Techniques: Immunoenzymatic Assay, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: High Adenosine Extracellular Levels Induce Glioblastoma Aggressive Traits Modulating the Mesenchymal Stromal Cell Secretome

doi: 10.3390/ijms21207706

Figure Lengend Snippet: ADO modified the BM-MSC secretome affecting the U343MG proliferation, GMT traits and cell motility. ( A ) U343MG cells were grown in 80% U343MG culture medium + 20% BM-MSC medium (CTRL), CM obtained from untreated cells (CM-CTRL) or cells treated with ADO for 48 h. At the end of the treatments, cell proliferation was evaluated using the MTS assay. The data are expressed as the percentage versus the CTRL, which was set to 100%, and they are presented as the mean values ± SEM of three independent experiments, each performed in duplicate. ( B , C ) U343MG cells were treated as described above and, after 72 h of treatment, mRNA expression levels of Slug, Snail, Twist, ZEB1, CDH1 and Vimentin were determined by RT-PCR. The data are expressed as fold changes with respect to untreated cells set to 1 and are the mean values ± SEM of two independent experiments. ( D , E ) U343MG cells were treated as above, and representative images ( D ) of the scratch wounds at 0 and 24 h after treatment are reported. ( E ) The data are expressed as percentage of gap closure after 24 h of treatment compared to the CTRL set to 100%. The data are represented as the means ± SEM of at least two independent experiments performed in triplicate. The significance of the differences was determined by one-way ANOVA, followed by Bonferroni’s post-hoc test: # p < 0.05, ## p < 0.01 vs. CTRL; * p < 0.05 vs. CM-CTRL.

Article Snippet: Human bone marrow MSCs (BM-MSCs) and glioblastoma stem cells (U343MG) were purchased by CLS (CLS Cell Lines Service GmbH, Eppelheim, Germany).

Techniques: Modification, MTS Assay, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: High Adenosine Extracellular Levels Induce Glioblastoma Aggressive Traits Modulating the Mesenchymal Stromal Cell Secretome

doi: 10.3390/ijms21207706

Figure Lengend Snippet: Effects of ADO in the BM-MSC and GBM cell cross-talk. ( A ) The image depicts the contact-independent transwell cultures with BM-MSCs in the upper chamber and U343MG in the lower chamber. U343MG cell proliferation was evaluated after 24 and 48 h with Crystal violet, as reported in . ( B ) The image depicts the contact-independent transwell cultures with U343MG in the upper chamber and BM-MSCs in the lower chamber. BM-MSCs cell proliferation was evaluated after 48 h with Crystal violet, as reported in . ( C ) U343MG cells were seeded in the upper chamber and BM-MSCs treated with ADO (100 nM and 100 µM) in the lower chamber. After 24 h, U343MG cell invasion was analyzed using Matrigel basement membrane transwell system, as described in . Representative images are shown. Cell migration was quantified by counting the number of cells that migrated into the lower surface of the transwell membrane. The data are reported as percentage of cell invasion versus the CTRL set to 100% and are the means ± SEM of three independent experiments. The significance of the differences was determined by one-way ANOVA, followed by Bonferroni’s post-hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. CTRL.

Article Snippet: Human bone marrow MSCs (BM-MSCs) and glioblastoma stem cells (U343MG) were purchased by CLS (CLS Cell Lines Service GmbH, Eppelheim, Germany).

Techniques: Migration

Journal: Scientific Reports

Article Title: Cell-specific pattern of berberine pleiotropic effects on different human cell lines

doi: 10.1038/s41598-018-28952-3

Figure Lengend Snippet: Intracellular localization of berberine and effects on viability in HDF, U343 and MIA PaCa-2 cells. ( a ) Confocal images of berberine distribution in HDF, U343 and MIA PaCa-2 cells. Cells were photographed 1 hour after treatment with berberine (10 μM, 50 μM or 150 μM). Black arrows point out nuclei. Scale bars represent 5 μm. ( b ) Reduction of cell viability after 48 hours of treatments with 0.4 μM, 2 μM, 10 μM, 50 μM berberine in HDF, U343 and MIA PaCa-2 cells. Graph columns represent mean of viable cells ± S.D. normalized versus control group (DMSO). *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: We used the human glioblastoma U343 GBM cells (U-343 MGa, CLS Cell Lines Service), the pancreatic carcinoma MIA PaCa-2 cells and normal primary dermal fibroblasts HDF (American Type Culture Collection ATCC, USA).

Techniques:

Journal: Scientific Reports

Article Title: Cell-specific pattern of berberine pleiotropic effects on different human cell lines

doi: 10.1038/s41598-018-28952-3

Figure Lengend Snippet: Berberine localizes in mitochondria and affects mitochondrial function. ( a ) Berberine was visualized by confocal microscopy in mitochondria of HDF, U343 and MIA PaCa-2 cells after 48 hours of exposure to 10 μM or 50 μM berberine. Merge columns represent overlapping of the berberine green signal with the TMRM red signal. DMSO-treated cells, used as a control, lack green fluorescence. Differential interference contrast (DIC) highlighted the cell morphology. Scale bars indicate 5 μm. ( b ) Citrate synthase activity was measured in the three cell lines after treatments in the presence or absence of berberine as described in Methods. U = Units of enzymatic activity. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: We used the human glioblastoma U343 GBM cells (U-343 MGa, CLS Cell Lines Service), the pancreatic carcinoma MIA PaCa-2 cells and normal primary dermal fibroblasts HDF (American Type Culture Collection ATCC, USA).

Techniques: Confocal Microscopy, Fluorescence, Activity Assay

Journal: Scientific Reports

Article Title: Cell-specific pattern of berberine pleiotropic effects on different human cell lines

doi: 10.1038/s41598-018-28952-3

Figure Lengend Snippet: Berberine alters the cell cycle and the transcriptional profile of cell cycle regulators. ( a ) Representative images of cell cycle distribution of HDF, U343 and MIA PaCa-2 cells. Cell cycle distribution was assayed by flow cytometry, following treatment with 10 μM or 50 μM berberine for 48 hours; subG1 (yellow bars), G1 (blue bars), S (green bars), G2 (red bars). Upper right insets represent the zoomed in subG1 population relative to each graph. Cell number (count) is reported in y-axis; fluorescence intensity (DsRed/PE) is reported in x-axis. ( b ) Quantification of cell cycle distribution; ( c ) The transcriptional profile of P53 , P21 , P16 was analyzed by Real Time RT-PCR after 1 hour or 48 hours berberine treatments. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: We used the human glioblastoma U343 GBM cells (U-343 MGa, CLS Cell Lines Service), the pancreatic carcinoma MIA PaCa-2 cells and normal primary dermal fibroblasts HDF (American Type Culture Collection ATCC, USA).

Techniques: Flow Cytometry, Fluorescence, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Cell-specific pattern of berberine pleiotropic effects on different human cell lines

doi: 10.1038/s41598-018-28952-3

Figure Lengend Snippet: Berberine induces cell senescence in HDF, U343 and MIA PaCa-2 cells. ( a ) Brightfield images of SA β-gal-positive HDF, U343 and MIA PaCa-2 cells after berberine treatments. Treatments: 10 μM or 50 μM berberine or DMSO for 48 hours. Scale bars indicate 50μm. ( b ) The graph shows the number of SA β-gal-positive cells normalized to the control (DMSO). *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: We used the human glioblastoma U343 GBM cells (U-343 MGa, CLS Cell Lines Service), the pancreatic carcinoma MIA PaCa-2 cells and normal primary dermal fibroblasts HDF (American Type Culture Collection ATCC, USA).

Techniques:

Journal: Scientific Reports

Article Title: Cell-specific pattern of berberine pleiotropic effects on different human cell lines

doi: 10.1038/s41598-018-28952-3

Figure Lengend Snippet: Effects of berberine on caspase-3 activity. ( a ) HDF, U343 and MIA PaCa-2 cells were incubated for 48 hours with 50 μM berberine and analyzed for caspase-3 activity monitored for 5 hours. Caspase-3 activity was measured as absorbance variation/hour/mg protein. Ber = berberine. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: We used the human glioblastoma U343 GBM cells (U-343 MGa, CLS Cell Lines Service), the pancreatic carcinoma MIA PaCa-2 cells and normal primary dermal fibroblasts HDF (American Type Culture Collection ATCC, USA).

Techniques: Activity Assay, Incubation

Journal: Scientific Reports

Article Title: Cell-specific pattern of berberine pleiotropic effects on different human cell lines

doi: 10.1038/s41598-018-28952-3

Figure Lengend Snippet: Berberine induces autophagy in U343 and MIA PaCa-2 cells, but not in HDF. ( a ) Relative mRNA expression is reported in y-axis. The treatments with berberine or DMSO alone are in x-axis. The values reported in graphs represent the mean ± S.D. ( b ) Immunofluorescence experiments. Cells were treated with berberine or DMSO for 48 hours and immunohistochemistry was performed with LC3 antibody (red fluorescence), as described in Methods. Nuclei are visualized by Hoechst staining. ( c ) Cells were treated as in ( b ) and proteins were subjected to electrophoresis and western blotting as described in Methods. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: We used the human glioblastoma U343 GBM cells (U-343 MGa, CLS Cell Lines Service), the pancreatic carcinoma MIA PaCa-2 cells and normal primary dermal fibroblasts HDF (American Type Culture Collection ATCC, USA).

Techniques: Expressing, Immunofluorescence, Immunohistochemistry, Fluorescence, Staining, Electrophoresis, Western Blot

Journal: Scientific Reports

Article Title: Cell-specific pattern of berberine pleiotropic effects on different human cell lines

doi: 10.1038/s41598-018-28952-3

Figure Lengend Snippet: Wound healing and cell invasion assays. ( a ) Representative brightfield images of the scratches (marked by white lines) in MIA PaCa-2 cells at 0, 24 and 48 hours, after treatments with berberine or DMSO. ( b ) Representative brightfield images of the scratches in U343 cells at 0, 24, 48 and 72 hours, after treatments with berberine or DMSO. ( c , d ) The values reported in the graphs represent the mean distance taken at each time from the wound edges (normalized to the DMSO group) ±S.D. Ber = berberine. ( e ) Impaired invasion of berberine-treated MIA PaCa-2 in transwell assay. ( f ) The graph reports the relative invasive score (±S.D.) corresponding to each berberine concentration, compared to DMSO. ( g ) The transcriptional profile of DAP1 and CXCR4 was analyzed by Real Time RT-PCR. The treatments with berberine or DMSO alone are in x-axis. The values reported in graphs represent the mean ± S.D. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: We used the human glioblastoma U343 GBM cells (U-343 MGa, CLS Cell Lines Service), the pancreatic carcinoma MIA PaCa-2 cells and normal primary dermal fibroblasts HDF (American Type Culture Collection ATCC, USA).

Techniques: Transwell Assay, Concentration Assay, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Cell-specific pattern of berberine pleiotropic effects on different human cell lines

doi: 10.1038/s41598-018-28952-3

Figure Lengend Snippet: Effects of berberine on the transcriptional profile of DNMT1, DNMT3A, DNMT3B and MGMT in HDF, U343 and MIA PaCa-2 cells. Cells were treated with berberine for 1 hour or 48 hours and Real Time RT-PCR was performed as described in Methods. The treatments with berberine or DMSO alone are in x-axis. The values reported in graphs represent the mean ± S.D. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: We used the human glioblastoma U343 GBM cells (U-343 MGa, CLS Cell Lines Service), the pancreatic carcinoma MIA PaCa-2 cells and normal primary dermal fibroblasts HDF (American Type Culture Collection ATCC, USA).

Techniques: Quantitative RT-PCR