Journal: Animal Cells and Systems

Article Title: Combined inhibition of STAT and Notch signalling effectively suppresses tumourigenesis by inducing apoptosis and inhibiting proliferation, migration and invasion in glioblastoma cells

doi: 10.1080/19768354.2021.1942983

Figure Lengend Snippet: Activation of Jagged/Notch and STATs in different glioblastoma cell lines and expression correlation of different STATs with Jagged1 in glioblastoma patients. (A) Expression correlation of STAT3, STAT5A and STAT5B with Jagged1 was performed using a TCGA dataset pool of 530 GBM patients. STAT3 ( R = 0.348, P < 0.001), STAT5A ( R = 0.187, P < 0.001) and STAT5B ( R = 0.347, P < 0.001) expression levels significantly correlated with those of Jagged1 in human glioblastoma patients. (B) Western blot analysis of Jagged1, NICD, p-STAT3 and p-STAT5 protein levels in a panel of human glioblastoma cell lines. An astrocyte cell line was used as a normal cell control and GAPDH was used as the loading control.

Article Snippet: Human glioblastoma cell lines (LN18, A172, LN229, and U87MG) and the astrocyte cell line were purchased from the American Type Culture Collection (ATCC).

Techniques: Activation Assay, Expressing, Western Blot, Control

Journal: Animal Cells and Systems

Article Title: Combined inhibition of STAT and Notch signalling effectively suppresses tumourigenesis by inducing apoptosis and inhibiting proliferation, migration and invasion in glioblastoma cells

doi: 10.1080/19768354.2021.1942983

Figure Lengend Snippet: STAT inhibitors induce Notch signalling in glioblastoma cells. A panel of glioblastoma cell lines (LN18, LN18-EGFRvIII, LN229, A172, U87MG-EGFRvIII) were checked for their Jagged1 and NICDs levels by Western blotting 24 h post-treatment in (A) with PMZ (15 µM) and (C) with S3I-201 (100–300 µM). Vehicle only (DMSO) was used as control. GAPDH served as a loading control. Relative cellular mRNA levels for Jagged1, Notch1 and Notch target genes (Hes1, Hey1, Hey2, Hrt2) were quantified by RT-qPCR in LN18 and LN18-EGFRvIII cells treated in (B) with PMZ (15 µM) and (D) with S3I-201 (300 µM) 24 h post-treatment. DMSO only treatment was used as control, with relative expression defined as 1.0. Individual samples in the graphical data are shown with mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, using the Student’s unpaired t -test.

Article Snippet: Human glioblastoma cell lines (LN18, A172, LN229, and U87MG) and the astrocyte cell line were purchased from the American Type Culture Collection (ATCC).

Techniques: Western Blot, Control, Quantitative RT-PCR, Expressing

Journal: Oncology Letters

Article Title: miR-9-3p inhibits glioma cell proliferation and apoptosis by directly targeting FOXG1

doi: 10.3892/ol.2020.11725

Figure Lengend Snippet: Effects of miR-9-3p overexpression on the proliferation of U87 cells. U87 cells were transfected with miR-9-3p mimic and NC mimic (50 pmol/ml) for 24 h. (A and C) RT-qPCR was applied to quantify the expression level of miR-9-3p in U87 MG and TG-905 cells. (B and D) The cell viability of U87 cells was determined by a CCK-8 assay in U87 MG and TG-905 cells. ##P<0.01 vs. NC mimic group. miR-9-3p, microRNA-9-3p; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; NC, negative control; control, without any treatment.

Article Snippet: Cell culture and transfection The human glioblastoma U87 MG (CL-0238) and TG-905 (CL-0309) cell lines were purchased from Procell Life Science & Technology Co., Ltd. (the origin of glioblastoma U87 MG cell is unknown and the cell line is preserved at the ATCC).

Techniques: Over Expression, Transfection, Quantitative RT-PCR, Expressing, CCK-8 Assay, Real-time Polymerase Chain Reaction, Negative Control

Journal: Oncology Letters

Article Title: miR-9-3p inhibits glioma cell proliferation and apoptosis by directly targeting FOXG1

doi: 10.3892/ol.2020.11725

Figure Lengend Snippet: Effects of miR-9-3p overexpression on the apoptosis of U87 cells. U87 cells were transfected with miR-9-3p mimic and NC mimic (50 pmol/ml) for 24 h. (A) Annexin-V/PI double-staining assay was applied to detect the cell apoptotic rate in U87 MG cells. (B) Annexin-V/PI double-staining assay was applied to detect the cell apoptotic rate in TG-905 cells. ##P<0.01 vs. NC mimic group. miR-9-3p, microRNA-9-3p; NC, negative control; PI, propidium iodide; control, without any treatment.

Article Snippet: Cell culture and transfection The human glioblastoma U87 MG (CL-0238) and TG-905 (CL-0309) cell lines were purchased from Procell Life Science & Technology Co., Ltd. (the origin of glioblastoma U87 MG cell is unknown and the cell line is preserved at the ATCC).

Techniques: Over Expression, Transfection, Double Staining, Negative Control

Journal: Oncology Letters

Article Title: miR-9-3p inhibits glioma cell proliferation and apoptosis by directly targeting FOXG1

doi: 10.3892/ol.2020.11725

Figure Lengend Snippet: FOXG1 is a direct target of miR-9-3p in glioma cells. (A) Bioinformatics analysis of the predicted interactions of miR-9-3p and its binding sites within the 3′-UTR of FOXG1. (B) Relative luciferase activities of FOXG1-wt, and FOXG1-mut were identified using a Dual-Luciferase Reporter assay kit following transfection of U87 MG with miR-9-3p mimic and NC mimic (50 pmol/ml) for 24 h. (C) The expression level of FOXG1 mRNA was examined by RT-qPCR following transfection of U87 MG with miR-9-3p mimic and NC mimic (50 pmol/ml) for 24 h. (D) The expression level of FOXG1 protein was examined by western blot assay following transfection of U87 MG with miR-9-3p mimic and NC mimic (50 pmol/ml) for 48 h. The relative intensity of FOXG1 protein is presented as a bar graph. (E) Relative luciferase activities of FOXG1-wt, and FOXG1-mut were identified using a Dual-Luciferase Reporter assay kit following transfection of TG-905 cells with miR-9-3p mimic and NC mimic (50 pmol/ml) for 24 h. (F) The expression level of FOXG1 mRNA was examined by RT-qPCR following transfection of TG-905 cells with miR-9-3p mimic and NC mimic (50 pmol/ml) for 24 h. (G) The expression level of FOXG1 protein was examined by western blot assay following transfection of TG-905 cells with miR-9-3p mimic and NC mimic (50 pmol/ml) for 48 h. The relative intensity of FOXG1 protein is presented as a bar graph. *P<0.05, **P<0.01 vs. NC mimic group. FOXG1, forkhead box G1; miR-9-3p, microRNA-9-3p; 3′-UTR, 3′-untranslated region; wt, wild-type; mut, mutant; NC, negative control.

Article Snippet: Cell culture and transfection The human glioblastoma U87 MG (CL-0238) and TG-905 (CL-0309) cell lines were purchased from Procell Life Science & Technology Co., Ltd. (the origin of glioblastoma U87 MG cell is unknown and the cell line is preserved at the ATCC).

Techniques: Binding Assay, Luciferase, Reporter Assay, Transfection, Expressing, Quantitative RT-PCR, Western Blot, Mutagenesis, Negative Control

Journal: Oncology Letters

Article Title: miR-9-3p inhibits glioma cell proliferation and apoptosis by directly targeting FOXG1

doi: 10.3892/ol.2020.11725

Figure Lengend Snippet: miR-9-3p regulates cell proliferation by inhibiting FOXG1. (A and C) Western blotting was used to detect the protein expression of FOXG1 following transfection of U87 MG and TG-905 cells with FOXG1 siRNA (40 pmol/ml) for 48 h. (B and D) Relative cell viability was determined by a Cell Counting Kit-8 assay following transfection of U87 MG and TG-905 cells with FOXG1 (40 pmol/ml) for 24 h. **P<0.01 vs. control group; ##P<0.01 vs. miR-9-3p mimic group; &&P<0.01 vs. FOXG1-siRNA group. FOXG1, forkhead box G1; miR-9-3p, microRNA-9-3p; siRNA, small interfering RNA; control, without any treatment.

Article Snippet: Cell culture and transfection The human glioblastoma U87 MG (CL-0238) and TG-905 (CL-0309) cell lines were purchased from Procell Life Science & Technology Co., Ltd. (the origin of glioblastoma U87 MG cell is unknown and the cell line is preserved at the ATCC).

Techniques: Western Blot, Expressing, Transfection, Cell Counting, Small Interfering RNA

Journal: Oncology Letters

Article Title: miR-9-3p inhibits glioma cell proliferation and apoptosis by directly targeting FOXG1

doi: 10.3892/ol.2020.11725

Figure Lengend Snippet: miR-9-3p regulates cell apoptosis by inhibiting FOXG1. (A) Annexin-V/PI double-staining assay was performed to detect the cell apoptotic rate of U87 MG cells following transfection with FOXG1 siRNA (40 pmol/ml) for 24 h. (B) Annexin-V/PI double-staining assay was applied to detect the cell apoptotic rate of TG-905 cells following transfection with FOXG1 siRNA (40 pmol/ml) for 24 h. **P<0.01 vs. control group; ##P<0.01 vs miR-9-3p mimic group; &&P<0.01 vs. FOXG1-siRNA group. miR-9-3p, microRNA-9-3p; FOXG1, forkhead box G1; PI, propidium iodide; siRNA, small interfering RNA; control, without any treatment.

Article Snippet: Cell culture and transfection The human glioblastoma U87 MG (CL-0238) and TG-905 (CL-0309) cell lines were purchased from Procell Life Science & Technology Co., Ltd. (the origin of glioblastoma U87 MG cell is unknown and the cell line is preserved at the ATCC).

Techniques: Double Staining, Transfection, Small Interfering RNA

Journal: International Journal of Molecular Sciences

Article Title: Suppression of Proliferation of Human Glioblastoma Cells by Combined Phosphodiesterase and Multidrug Resistance-Associated Protein 1 Inhibition

doi: 10.3390/ijms22189665

Figure Lengend Snippet: Antiproliferative activity of PDE inhibitors in glioblastoma cells. The heatmap illustrates the percentage of viable U87MG, A172 and T98G cells determined in the CCK-8 assay following treatment with PDE inhibitors at the indicated concentrations for 72 h ( n = 3–12). Detailed quantitative data are presented in .

Article Snippet: The human glioblastoma astrocytoma cell lines U87MG (ECACC 89081402), derived from a female patient with grade IV glioma [ ], and A172 (ECACC 88062428), derived from a 53-year-old male with glioblastoma [ ], were obtained from the European Collection of Authenticated Cell Cultures.

Techniques: Activity Assay, CCK-8 Assay

Journal: International Journal of Molecular Sciences

Article Title: Suppression of Proliferation of Human Glioblastoma Cells by Combined Phosphodiesterase and Multidrug Resistance-Associated Protein 1 Inhibition

doi: 10.3390/ijms22189665

Figure Lengend Snippet: Relative expression levels of genes encoding PDEs, adenylyl cyclases and guanylyl cyclases in the U87MG, A172 and T98G glioblastoma cells. ( A ) Representative gels illustrating mRNA expression levels in each of the three glioblastoma lines tested. White asterisk ( * ) on the gel indicates the band of the correct size. ( B ) mRNA expression levels normalised by the GAPDH mRNA signal in glioblastoma cells grown to maximal confluence. Data are presented as the mean ± standard deviation of three independent experiments.

Article Snippet: The human glioblastoma astrocytoma cell lines U87MG (ECACC 89081402), derived from a female patient with grade IV glioma [ ], and A172 (ECACC 88062428), derived from a 53-year-old male with glioblastoma [ ], were obtained from the European Collection of Authenticated Cell Cultures.

Techniques: Expressing, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Suppression of Proliferation of Human Glioblastoma Cells by Combined Phosphodiesterase and Multidrug Resistance-Associated Protein 1 Inhibition

doi: 10.3390/ijms22189665

Figure Lengend Snippet: Effects of combinations of PF-2545920 with other PDE inhibitors on the survival of glioblastoma cells. CCK-8 proliferation assays were performed in the U87MG ( A ), T98G ( B ) and A172 ( C ) cell lines cultured with 10 μM PF-2545920 (yellow bar) with other PDE inhibitors used either at a concentration that inhibited cell viability by not more than 20% or at the default concentration of 100 μM (blue bars), or with combinations of these inhibitors at the same concentration with 10 μM PF-2545920 (green bars). Data are presented as the mean ± standard deviation. The statistical significance of differences in cell survival in the presence of drug combinations is indicated as follows: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 (from survival under PDE inhibitor alone); & p < 0.05; && p < 0.01; &&& p < 0.001; &&&& p < 0.0001 (from survival under 10 μM PF-2545920).

Article Snippet: The human glioblastoma astrocytoma cell lines U87MG (ECACC 89081402), derived from a female patient with grade IV glioma [ ], and A172 (ECACC 88062428), derived from a 53-year-old male with glioblastoma [ ], were obtained from the European Collection of Authenticated Cell Cultures.

Techniques: CCK-8 Assay, Cell Culture, Concentration Assay, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Suppression of Proliferation of Human Glioblastoma Cells by Combined Phosphodiesterase and Multidrug Resistance-Associated Protein 1 Inhibition

doi: 10.3390/ijms22189665

Figure Lengend Snippet: Synergistic suppressive action of PF-2545920 and MY-5445 on the viability and migration of glioblastoma cells. ( A ) Concentration–response relationships for the inhibitory action of PF-2545920 on the viability of U87MG, A172 and T98G cells were obtained using the CCK-8 assay in the absence or presence of MY-5445 at 25–100 μM. ( B ) In the synergy plots, the colour indicates the degree of synergism, and the values indicate synergy scores using the Loewe model as previously described . The stronger the synergy is, the darker blue is the background, and the higher is the synergy score. Asterisks indicate the significance of synergy scores obtained following a one sample t -test (* p < 0.05; ** p <0.001, *** p < 0.0001; the number of replicates (N) is shown in the left top corner of the matrix display). ( C ) Migration capability of U87MG, A172 and T98G cells was assessed from the degree of closure of the initial cell-free gap area (outlined by a contour line in the centre of each image) after 24 h in culture using the Radius™ cell migration assay. C: control (vehicle); MY: 50 μM MY-5445; PF: 10 μM PF-2545920: PF+MY: combination treatment. Concentrations for the migration experiment were chosen from the synergy points designated by ovals in ( B ). Data are presented as the mean ± standard deviation ( n = 3). The statistical significance of differences in cell migration is indicated as follows: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 (post hoc Tukey’s test).

Article Snippet: The human glioblastoma astrocytoma cell lines U87MG (ECACC 89081402), derived from a female patient with grade IV glioma [ ], and A172 (ECACC 88062428), derived from a 53-year-old male with glioblastoma [ ], were obtained from the European Collection of Authenticated Cell Cultures.

Techniques: Migration, Concentration Assay, CCK-8 Assay, Cell Migration Assay, Control, Standard Deviation

Journal: Cell Death & Disease

Article Title: Potent effect of the MDM2 inhibitor AMG232 on suppression of glioblastoma stem cells

doi: 10.1038/s41419-018-0825-1

Figure Lengend Snippet: a Chemical structures of RG7112 and AMG232. b , c Cell numbers of U373MG, LN18, U251MG, A1207, DBTRG-05MG, and U87MG cells treated with different concentrations (0.07–54 μM) of RG7112 or AMG232 for 72 h were evaluated by quantifying image-based cell counting. Nonlinear regression analyses of dose−response curves are shown ( n = 3). Error bars represent standard deviation (SD). d The IC 50 values obtained from dose−response curves are shown. Each bars represent mean ( n = 3) and 95% confidence interval (CI). e IC 50 values of TP53 mutant cell lines ( n = 3) and TP53 wild-type cell lines ( n = 3) to RG7112 and AMG232 are shown. Data represent the mean and SD error (* p < 0.01). f An image-based immunofluorescence assay for p21 (green), p53 (red) and DAPI (blue) in A1207 cells treated with DMSO, RG7112 (2 μM), AMG232 (2 μM), and Camptothecin (10 μM) for 72 h are shown. g p21 fluorescence intensity measurements for A1207 cells treated with increasing concentrations of RG7112 or AMG232 for 72 h are shown. The Z-factor for 18 μM RG7112 and 18 μM AMG232 in A1207 cells are indicated. h The mean percentage of p21-positive cells ( n = 3, SD) for six glioblastoma cell lines treated with DMSO, RG7112 (2 μM), AMG232 (2 μM) for 72 h are shown

Article Snippet: Three TP53 mutant glioblastoma cell lines (U373MG, LN18, and U251MG) and three TP53 wild-type glioblastoma cell lines (A1207, DBTRG-05MG, and U87MG) were either purchased from the American Type Culture Collection (ATCC) or kindly provided by Prof. Shin-Hyuk Kang at Korea University, Seoul, Korea.

Techniques: Cell Counting, Standard Deviation, Mutagenesis, Immunofluorescence, Fluorescence

Journal: Cell Death & Disease

Article Title: Potent effect of the MDM2 inhibitor AMG232 on suppression of glioblastoma stem cells

doi: 10.1038/s41419-018-0825-1

Figure Lengend Snippet: a U138MG, LN18, and A1207 cells were treated with RG7112 for 12 h at the indicated concentrations. p21, MDM2, p53, and PARP levels were examined by immunoblotting. Actin was used as a loading control. b A1207 cells were transfected with control or p53 siRNA for 48 h prior to treatment with DMSO or 1 μM AMG232 for 12 h. The levels of p53 and p21 were examined by immunoblotting. Actin was used as a loading control. c A1207 cells transfected with control or p53 siRNA for 48 h were treated with different concentrations (0.014–10.8 μM) of RG7112 or AMG232 for 72 h were evaluated by quantifying image-based cell counting. Nonlinear regression analyses of dose−response curves are shown ( n = 3, SD error). d The graph represents IC 50 values comparing control and p53-depleted A1207 cells for RG7112 and AMG232. Data show mean and SD error (* p < 0.01)

Article Snippet: Three TP53 mutant glioblastoma cell lines (U373MG, LN18, and U251MG) and three TP53 wild-type glioblastoma cell lines (A1207, DBTRG-05MG, and U87MG) were either purchased from the American Type Culture Collection (ATCC) or kindly provided by Prof. Shin-Hyuk Kang at Korea University, Seoul, Korea.

Techniques: Western Blot, Control, Transfection, Cell Counting

Journal: Cell Death & Disease

Article Title: Potent effect of the MDM2 inhibitor AMG232 on suppression of glioblastoma stem cells

doi: 10.1038/s41419-018-0825-1

Figure Lengend Snippet: a An experiment design of high content analysis for patient-derived glioblastoma stem cells are represented. Ten patient-derived glioblastoma stem cells were isolated and grown as neurosphere as shown in upper left panel. Stem cells were cultured in laminin-coated 384-well plates (upper right panel) were treated with RG7112 and AMG232 (0.7 nM−50 μM) for 72 h and were analyzed by automated microscopy for image-based cell counting and p21 immunofluorescence assay. Representative images of p21 (green) and DAPI (blue) in 526T stem cells treated with DMSO, RG7112 (1.8 μM), AMG232 (1.8 μM) for 72 h are shown in lower panel. b p21 fluorescence intensity measurements for DMSO, RG7112 (1.8 μM), and AMG232 (1.8 μM)-treated cells ( n = 3). The gray line indicates the threshold (Z-score = 1.5). c The mean percentage of p21-positive cells for ten patient-derived glioblastoma stem cells treated with DMSO, RG7112 (1.8 μM), and AMG232 (1.8 μM) for 72 h are shown. Data represent mean ( n = 3) and SD error. d IC 50 values comparing RG7112 and AMG232 in ten patient-derived glioblastoma stem cells obtained from dose−response curves are shown ( n = 3, CI error, * p < 0.05, ** p < 0.01). Nonlinear regression analysis for these data are shown in Supplementary Figure 6

Article Snippet: Three TP53 mutant glioblastoma cell lines (U373MG, LN18, and U251MG) and three TP53 wild-type glioblastoma cell lines (A1207, DBTRG-05MG, and U87MG) were either purchased from the American Type Culture Collection (ATCC) or kindly provided by Prof. Shin-Hyuk Kang at Korea University, Seoul, Korea.

Techniques: High Content Screening, Derivative Assay, Isolation, Cell Culture, Microscopy, Cell Counting, Immunofluorescence, Fluorescence

Journal: Cell Death & Disease

Article Title: Potent effect of the MDM2 inhibitor AMG232 on suppression of glioblastoma stem cells

doi: 10.1038/s41419-018-0825-1

Figure Lengend Snippet: a Location of TP53 gene mutation in six patient-derived glioblastoma stem cells. Green circles represent missense mutations and a black circle indicates a splice-site mutation. The amino acid change is indicated above each circle and the name of patient-derived glioblastoma stem cells is bracketed. b The graph shows MDM2 gene copy numbers in ten patient-derived glioblastoma stem cells. P value is indicated in the graph. c Scatter plots showing IC 50 values of patient-derived glioblastoma stem cells to RG7112 and AMG232. The stem cells are classified according to TP53 mutation status. P value is indicated in the graph. d A map of gene-high content analysis association. Genetic status of TP53 , MDM2 , and IC 50 , p21 fold-changes of the MDM2 inhibitors in ten patient-derived glioblastoma cells are summarized. Color scales with IC 50 and Z-score values are indicated. e 526T, 578T, and 775T stem cells were treated with RG7112 and AMG232 for 72 h at the indicated concentrations. p21, MDM2, and p53 levels were evaluated by immunoblotting. Actin was used as a loading control

Article Snippet: Three TP53 mutant glioblastoma cell lines (U373MG, LN18, and U251MG) and three TP53 wild-type glioblastoma cell lines (A1207, DBTRG-05MG, and U87MG) were either purchased from the American Type Culture Collection (ATCC) or kindly provided by Prof. Shin-Hyuk Kang at Korea University, Seoul, Korea.

Techniques: Mutagenesis, Derivative Assay, High Content Screening, Western Blot, Control

Journal: Cell Death & Disease

Article Title: Potent effect of the MDM2 inhibitor AMG232 on suppression of glioblastoma stem cells

doi: 10.1038/s41419-018-0825-1

Figure Lengend Snippet: a Three-dimensional (3D) tumor spheroids of 526T, 578T, and 775T patient-derived glioblastoma cells were treated with indicated concentrations of RG7112 and AMG232. Representative images show the response of 3D spheroids to RG7112 and AMG232 at day 14. b The sphere images were taken at 2-day intervals up to 14 days and the sizes were measured and analyzed. Data represent the mean ( n = 3) and SD error. c 578T cells were treated with 0.1 μM AMG232 for 72 h and analyzed by immunoblotting (see Supplementary Figure ). The steady-state levels of Nestin and ZEB1 relative to Actin were quantified. Data show the mean and error ( n = 2, SD, * p < 0.05)

Article Snippet: Three TP53 mutant glioblastoma cell lines (U373MG, LN18, and U251MG) and three TP53 wild-type glioblastoma cell lines (A1207, DBTRG-05MG, and U87MG) were either purchased from the American Type Culture Collection (ATCC) or kindly provided by Prof. Shin-Hyuk Kang at Korea University, Seoul, Korea.

Techniques: Derivative Assay, Western Blot