tp53 wild type glioblastoma cell lines  (ATCC)

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