u87 idh1 r132h (ATCC)
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U87 Idh1 R132h, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 99 stars, based on 1 article reviews
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Images
1) Product Images from "Targeting IDH1/2 mutant cancers with combinations of ATR and PARP inhibitors"
Article Title: Targeting IDH1/2 mutant cancers with combinations of ATR and PARP inhibitors
Journal: NAR Cancer
doi: 10.1093/narcan/zcab018
Figure Legend Snippet: IDH1 mutation confers sensitivity to ATR inhibitors. ( A–C ) Five-day short-term cell viability assays of four separate ATR inhibitors AZD6738, ATRIN175, VE-822 and BAY-1895344. U87 (WT and IDH1 R132H/+), HCT116 (WT and IDH1 R132H/+) and LN229 (WT and doxycycline inducible IDH1 R132H/+) cells were used for this assay. ( D ) IC 50 ratios of screened WT and the IDH1 mutant cells indicate that the IDH1 mutant cells are more sensitive to all the four ATR inhibitors tested. ( E ) HCT116 WT and R132H/+ cells were treated with AZD6738 for 14 days ( n = 6). Error bars represent means ± SEM, * P < 0.05.
Techniques Used: Mutagenesis
Figure Legend Snippet: ATR inhibitor increases PARP inhibitor mediated sensitivity in IDH1 mutant cells. ( A ) Quantification and representative images of clonogenic survival assays of HCT116 WT and IDH1-mutant (R132H/+) cells were treated with AZD6738 alone (solid lines) and in combination with 1 μM olaparib (dashed lines) for 14 days ( n = 6). ( B ) Quantification representative images of clonogenic survival assays of U87 WT and IDH1-mutant (R132H/+) cells were treated with AZD6738 alone (solid lines) and in combination with 2 μM olaparib (dashed lines) for 14 days ( n = 4). ( C ) Quantification of clonogenic survival assay of SW1353 (IDH2 R172S/+) cells treated with AZD6738 alone (blue line) and in combination with 1 μM olaparib (red line) for 14 days. ( D ) Quantification of clonogenic survival assay of RBE (IDH1 R132S/+) cells treated with AZD6738 alone (blue line) and in combination with 2 μM Olaparib (red line) for 14 days. Error bars represent means ± SEM. *** P < 0.001, ** P < 0.01, * P < 0.05.
Techniques Used: Mutagenesis, Clonogenic Cell Survival Assay
Figure Legend Snippet: ATR and PARP inhibition increases unrepaired damage and suppresses HR. ( A ) U87 WT and IDH1-R132H/+ cells were treated with olaparib (10 μM), AZD6738 (5 nM) or both for 4 and 24 h. Cell were fixed and stained for RPA70 and counterstained with DAPI. Cells with more than 20 RPA70 foci were counted in seven distinct fields. In total 250–350 cells were analyzed. Representative images of cells stained with RPA (green) and counterstained with DAPI (Blue) are shown. The images shown were acquired using a 40× objective lens. The scale bar is 10 μm. ( B ) HCT116 WT and IDH1-R132H/+ cells were treated with olaparib (1 μM), AZD6738 (500 nM) or both for 24 h. Cell were fixed and stained for γ-H2AX and counterstained with DAPI. Cells with more than 10 γ-H2AX foci were counted in eight distinct fields. In total 800–1000 cells were analyzed. Representative images of cells stained with γ-H2AX (green) and counterstained with DAPI (Blue) are shown. The images shown were acquired using a 40× objective lens. The scale bar is 10 μm. ( C ) Quantitation of ligand inducible U2OS-DR-GFP assay where cells were cultured with or without 300 μM 2HG for 3 days and with and without olaparib (1 μM), AZD6738 (500 nM) or both for 24 h prior to ligand induction ( n = 3). ( D ) Quantitation of control U2OS-DR-GFP assay without ligand induction. Dotplots show GFP population with and without ligand addition. Error bars represent means ± SEM. *** P < 0.001, ** P < 0.01, * P < 0.05.
Techniques Used: Inhibition, Staining, Quantitation Assay, Cell Culture
Figure Legend Snippet: ATR and PARP inhibition cause IDH1/2 mutants to prematurely enter mitosis. ( A and B ) HCT116 and U87 (WT and IDH1-R132H/+) cells were treated with olaparib (1μM), AZD6738 (500 nM) or both for 24 h. Cell were fixed and stained for p-H3 (Ser10) and counterstained with DAPI. Number of p-H3 positive cells were analyzed in 5 distinct fields ( n = 2). ( C ) Representative p-H3-Alexa-647 plots of HCT116 WT and R132H/+ cells that were treated with olaparib (1 μM), AZD6738 (500 nM) or both for 24 h. DNA content (propidium iodide) and pH3-Ser10/Alexa-488 were assessed by flow cytometry ( n = 3). Quantification of result shown on the right panel. ( D ) Representative EdU-Alexa-647 plots of HCT116 WT and R132H/+ cells were treated with olaparib (1 μM), AZD6738 (500 nM) or both for 24 h DNA content (propidium iodide) and EdU -Ser10/Alexa-488 were assessed by flow cytometry ( n = 3). ( E ) HCT116 (WT and IDH1-R132H/+) cells were treated with olaparib (1 μM), AZD6738 (500 nM) or both for 24 h. Cell were fixed and stained for cyclin-A and counterstained with DAPI. Number of cylin-A positive cells were analyzed in five distinct fields ( n = 3). Quantification of result shown on the right panel. Error bars represent means ± SEM. *** P < 0.001, ** P < 0.01, * P < 0.05.
Techniques Used: Inhibition, Staining, Flow Cytometry
Figure Legend Snippet: ATRi and PARPi combination caused increased apoptosis in IDH1-mutant cells. HCT116 (WT and IDH1-R132H/+) cells were treated with olaparib (1 μM), AZD6738 (500 nM) or both for 24 h (left panel) or 48 h (right panel). Lysates were harvested after 24 and 48 h. Cleaved caspase-3 and cleaved PARP was evaluated by western blot analysis.
Techniques Used: Mutagenesis, Western Blot
Figure Legend Snippet: Combination of ATRi and PRAPi was effective in IDH1-mutant mouse xenograft model. ( A ) Athymic nude mice received subcutaneous injection of HCT116 IDH1 R132H/+ or HT1080 IDH1 R132C/+ cells. Twelve days after injection, the hind flank tumors were measured and equally distributed to four-arm treatment groups ( B ) Left panel: Mice carrying flank tumors of HCT116 R132H/+ cells were treated with no treatment ( n = 8), Olaparib alone (50 mg/kg) ( n = 8), AZD6738 (50 mg/kg) ( n = 8), or Olaparib (50 mg/kg) and AZD6738 (50 mg/kg) ( n = 8). Mice were treated daily for 28 days. Mean tumor volume per group with SEM is plotted on y-axis. Right panel: mean body weight with SEM of mice during HCT116 IDH1 R132H/+ flank tumor experiment. ( C ) Left panel: mice carrying flank tumors of HT1080 'cells were treated with no treatment ( n = 7), Olaparib alone (50 mg/kg) ( n = 7), AZD6738 (50 mg/kg) ( n = 7), or Olaparib (50 mg/kg) and AZD6738 (50 mg/kg) ( n = 8). Mice were treated daily for 28 days. Right panel: mean body weight with SEM of mice during HT1080 flank tumor experiment. ( D ) Left panel: mice carrying flank tumors of HCT116 R132H/+ cells were treated with no treatment ( n = 5), Olaparib alone (25 mg/kg) ( n = 5), AZD6738 (25 mg/kg) ( n = 5), or Olaparib (25 mg/kg) and AZD6738 (25 mg/kg) ( n = 5). Mice were treated daily for 21 days. Mean tumor volume per group with SEM is plotted on y-axis. Right panel: mean body weight with SEM of mice during HCT116 R12H/+ flank tumor experiment. Error bars represent means ± SEM. P values were calculated using two-way ANOVA.
Techniques Used: Mutagenesis, Injection