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u87 idh1 r132h  (ATCC)


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    Structured Review

    ATCC u87 idh1 r132h
    <t>IDH1</t> 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. <t>U87</t> (WT and IDH1 <t>R132H/+),</t> 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.
    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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    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

    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.
    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

    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.
    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

    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.
    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

    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.
    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

    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.
    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

    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.
    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



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    Image Search Results


    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.

    Journal: NAR Cancer

    Article Title: Targeting IDH1/2 mutant cancers with combinations of ATR and PARP inhibitors

    doi: 10.1093/narcan/zcab018

    Figure Lengend 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.

    Article Snippet: The U87 IDH1 R132H/+ (ATCC ® HTB-14IG™), HT1080 IDH1 R132C (ATCC® CCL-121™) and SW1353 IDH2 R172S (ATCC ® HTB-94) cell lines was purchased from ATCC.

    Techniques: Mutagenesis

    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.

    Journal: NAR Cancer

    Article Title: Targeting IDH1/2 mutant cancers with combinations of ATR and PARP inhibitors

    doi: 10.1093/narcan/zcab018

    Figure Lengend 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.

    Article Snippet: The U87 IDH1 R132H/+ (ATCC ® HTB-14IG™), HT1080 IDH1 R132C (ATCC® CCL-121™) and SW1353 IDH2 R172S (ATCC ® HTB-94) cell lines was purchased from ATCC.

    Techniques: Mutagenesis, Clonogenic Cell Survival Assay

    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.

    Journal: NAR Cancer

    Article Title: Targeting IDH1/2 mutant cancers with combinations of ATR and PARP inhibitors

    doi: 10.1093/narcan/zcab018

    Figure Lengend 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.

    Article Snippet: The U87 IDH1 R132H/+ (ATCC ® HTB-14IG™), HT1080 IDH1 R132C (ATCC® CCL-121™) and SW1353 IDH2 R172S (ATCC ® HTB-94) cell lines was purchased from ATCC.

    Techniques: Inhibition, Staining, Quantitation Assay, Cell Culture

    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.

    Journal: NAR Cancer

    Article Title: Targeting IDH1/2 mutant cancers with combinations of ATR and PARP inhibitors

    doi: 10.1093/narcan/zcab018

    Figure Lengend 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.

    Article Snippet: The U87 IDH1 R132H/+ (ATCC ® HTB-14IG™), HT1080 IDH1 R132C (ATCC® CCL-121™) and SW1353 IDH2 R172S (ATCC ® HTB-94) cell lines was purchased from ATCC.

    Techniques: Inhibition, Staining, Flow Cytometry

    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.

    Journal: NAR Cancer

    Article Title: Targeting IDH1/2 mutant cancers with combinations of ATR and PARP inhibitors

    doi: 10.1093/narcan/zcab018

    Figure Lengend 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.

    Article Snippet: The U87 IDH1 R132H/+ (ATCC ® HTB-14IG™), HT1080 IDH1 R132C (ATCC® CCL-121™) and SW1353 IDH2 R172S (ATCC ® HTB-94) cell lines was purchased from ATCC.

    Techniques: Mutagenesis, Western Blot

    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.

    Journal: NAR Cancer

    Article Title: Targeting IDH1/2 mutant cancers with combinations of ATR and PARP inhibitors

    doi: 10.1093/narcan/zcab018

    Figure Lengend 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.

    Article Snippet: The U87 IDH1 R132H/+ (ATCC ® HTB-14IG™), HT1080 IDH1 R132C (ATCC® CCL-121™) and SW1353 IDH2 R172S (ATCC ® HTB-94) cell lines was purchased from ATCC.

    Techniques: Mutagenesis, Injection

    The expression of NDRG2 had an inverse association with pyruvate carboxylase in IDH1 (R132H)-mutant glioma cells. A. NDRG2 and pyruvate carboxylase immunostaining of tissue microarrays comprising IDH1 wild-type and IDH1 (R132H) glioma tissues with different differentiation states. Scale bar, 200 μm and 50 μm (magnification). B. Correlation analysis of the staining index for the expression of NDRG2 and PC proteins in the IDH1 wild-type glioma patient specimens (n = 27) and the IDH1-R132H mutant glioma patient specimens (n = 27). Pearson’s product-moment correlation coefficients and the P values are also shown. C. NDRG2 and pyruvate carboxylase expression in the IDH1 wild-type and IDH1 (R132H) glioma U87 cells. D. The relative protein levels of pyruvate carboxylase to β-actin were quantified by densitometry. *P<0.05, **P<0.01.

    Journal: American Journal of Cancer Research

    Article Title: NDRG2 inhibits pyruvate carboxylase-mediated anaplerosis and combines with glutamine blockade to inhibit the proliferation of glioma cells

    doi:

    Figure Lengend Snippet: The expression of NDRG2 had an inverse association with pyruvate carboxylase in IDH1 (R132H)-mutant glioma cells. A. NDRG2 and pyruvate carboxylase immunostaining of tissue microarrays comprising IDH1 wild-type and IDH1 (R132H) glioma tissues with different differentiation states. Scale bar, 200 μm and 50 μm (magnification). B. Correlation analysis of the staining index for the expression of NDRG2 and PC proteins in the IDH1 wild-type glioma patient specimens (n = 27) and the IDH1-R132H mutant glioma patient specimens (n = 27). Pearson’s product-moment correlation coefficients and the P values are also shown. C. NDRG2 and pyruvate carboxylase expression in the IDH1 wild-type and IDH1 (R132H) glioma U87 cells. D. The relative protein levels of pyruvate carboxylase to β-actin were quantified by densitometry. *P<0.05, **P<0.01.

    Article Snippet: The human glioma cell lines U251, T98G, IDH1 (R132H) U87, IDH1 (WT) U87 and HEK293T cells were purchased from ATCC and used in the present study.

    Techniques: Expressing, Mutagenesis, Immunostaining, Staining

    (A) TAGLN2 mRNA was expressed at significantly higher levels in IDH1/2 WT tumors compared to IDH1/2 mutant tumors in both our institutional (p-value=7.73×10 −5 ; FDR=0.053) and the validation TCGA (p-value <0.0001; FDR < 0.0001) cohorts. (B) Mass spectrometry identified higher TAGLN2 protein expression in IDH1/2 WT compared to IDH1/2 mutant LGG from our institutional cohort. Six different peptides corresponding to TAGLN2 protein were expressed at significantly up-regulated in IDH1/2 WT compared to IDH1/2 mutant tumors. (C) Publicly available TAGLN2 mRNA expression of all Grade II (G2, n=249), III (G3, n=265) and IV (GBM, n=153) gliomas from the TCGA database are significantly different (p<0.0001). (D) TAGLN2 expression in IDH1/2 WT Grade II (n=21), III (n=52) and IV (n=133) tumors from TCGA data was significantly different (p=5.136×10 −20 ).

    Journal: Oncotarget

    Article Title: Oncogenic transgelin-2 is differentially regulated in isocitrate dehydrogenase wild-type vs. mutant gliomas

    doi: 10.18632/oncotarget.26365

    Figure Lengend Snippet: (A) TAGLN2 mRNA was expressed at significantly higher levels in IDH1/2 WT tumors compared to IDH1/2 mutant tumors in both our institutional (p-value=7.73×10 −5 ; FDR=0.053) and the validation TCGA (p-value <0.0001; FDR < 0.0001) cohorts. (B) Mass spectrometry identified higher TAGLN2 protein expression in IDH1/2 WT compared to IDH1/2 mutant LGG from our institutional cohort. Six different peptides corresponding to TAGLN2 protein were expressed at significantly up-regulated in IDH1/2 WT compared to IDH1/2 mutant tumors. (C) Publicly available TAGLN2 mRNA expression of all Grade II (G2, n=249), III (G3, n=265) and IV (GBM, n=153) gliomas from the TCGA database are significantly different (p<0.0001). (D) TAGLN2 expression in IDH1/2 WT Grade II (n=21), III (n=52) and IV (n=133) tumors from TCGA data was significantly different (p=5.136×10 −20 ).

    Article Snippet: Since TAGLN2 was found to be expressed at significantly lower mRNA and protein levels in IDH1/2 mutant gliomas from both institutional and TCGA patient cohorts, we confirmed these findings in vitro by evaluating TAGLN2 protein levels in a commercially available U87 MG isogenic cell line overexpressing IDH1 R132H mutant protein (ATCC, Manassas, VA), which will be referred to as IDH1 mutant U87 MG cells.

    Techniques: Mutagenesis, Mass Spectrometry, Expressing

    (A) Promoter methylation was detected in IDH1/2 mutant tumors (n=54, cyan) and IDH1/2 WT tumors (n=8, salmon) from our institutional cohort using 15 CpG TAGLN2 promoter methylation sites included on the Illumina HM-450K array. IDH1/2 mutant showed significantly higher levels of methylation (FDR<0.05) in the majority of CpG islands corresponding to TAGLN2 (n=11), as demonstrated by the heat map. Low methylation levels are denoted in green and high methylation levels are denoted in red. (B) Methylation results were validated using methylation data from the publicly available TCGA cohort.

    Journal: Oncotarget

    Article Title: Oncogenic transgelin-2 is differentially regulated in isocitrate dehydrogenase wild-type vs. mutant gliomas

    doi: 10.18632/oncotarget.26365

    Figure Lengend Snippet: (A) Promoter methylation was detected in IDH1/2 mutant tumors (n=54, cyan) and IDH1/2 WT tumors (n=8, salmon) from our institutional cohort using 15 CpG TAGLN2 promoter methylation sites included on the Illumina HM-450K array. IDH1/2 mutant showed significantly higher levels of methylation (FDR<0.05) in the majority of CpG islands corresponding to TAGLN2 (n=11), as demonstrated by the heat map. Low methylation levels are denoted in green and high methylation levels are denoted in red. (B) Methylation results were validated using methylation data from the publicly available TCGA cohort.

    Article Snippet: Since TAGLN2 was found to be expressed at significantly lower mRNA and protein levels in IDH1/2 mutant gliomas from both institutional and TCGA patient cohorts, we confirmed these findings in vitro by evaluating TAGLN2 protein levels in a commercially available U87 MG isogenic cell line overexpressing IDH1 R132H mutant protein (ATCC, Manassas, VA), which will be referred to as IDH1 mutant U87 MG cells.

    Techniques: Methylation, Mutagenesis

    Clinical-pathological characteristics of patients analyzed for TAGLN2 mRNA expression in institutional and TCGA LGG cohorts

    Journal: Oncotarget

    Article Title: Oncogenic transgelin-2 is differentially regulated in isocitrate dehydrogenase wild-type vs. mutant gliomas

    doi: 10.18632/oncotarget.26365

    Figure Lengend Snippet: Clinical-pathological characteristics of patients analyzed for TAGLN2 mRNA expression in institutional and TCGA LGG cohorts

    Article Snippet: Since TAGLN2 was found to be expressed at significantly lower mRNA and protein levels in IDH1/2 mutant gliomas from both institutional and TCGA patient cohorts, we confirmed these findings in vitro by evaluating TAGLN2 protein levels in a commercially available U87 MG isogenic cell line overexpressing IDH1 R132H mutant protein (ATCC, Manassas, VA), which will be referred to as IDH1 mutant U87 MG cells.

    Techniques: Expressing, Mutagenesis

    Multi-variable analysis of clinical-pathological factors with OS from low(er) grade gliomas in the TCGA cohort

    Journal: Oncotarget

    Article Title: Oncogenic transgelin-2 is differentially regulated in isocitrate dehydrogenase wild-type vs. mutant gliomas

    doi: 10.18632/oncotarget.26365

    Figure Lengend Snippet: Multi-variable analysis of clinical-pathological factors with OS from low(er) grade gliomas in the TCGA cohort

    Article Snippet: Since TAGLN2 was found to be expressed at significantly lower mRNA and protein levels in IDH1/2 mutant gliomas from both institutional and TCGA patient cohorts, we confirmed these findings in vitro by evaluating TAGLN2 protein levels in a commercially available U87 MG isogenic cell line overexpressing IDH1 R132H mutant protein (ATCC, Manassas, VA), which will be referred to as IDH1 mutant U87 MG cells.

    Techniques:

    (A) GBM30 neurospheres stably expressing TAGLN2 shRNA and corresponding scrambled shRNA control were generated and the level of stable TAGLN2 knock-down detected by Western blot is shown. (B) GBM30 neurospheres with stable knock-down of TAGLN2 or scrambled shRNA control were counted at 24, 72, and 1120 hours after plating. Knock-down of TAGLN2 resulted in significantly decreased cell counts (p<0.05). (C) GBM30 neurospheres and (D) U87 MG glioma cells stably overexpressing TAGLN2 and corresponding vector control were generated and the level of stable TAGLN2 overexpression was detected by Western blot. Of note, endogenous TAGLN2 (22 Kda) and exogenous TAGLN2 -myc (28 kDa) are shown. (E) GBM30 neurospheres stably overexpressing TAGLN2 resulted in significantly increased cell proliferation compared to vector control at 72 and 120 hours (p<0.05). (F) U87 MG cells stably overexpressing TAGLN2 resulted in increased cell proliferation compared to the vector alone. Experiments were performed twice with six replicates each. * , statistically significant difference in proliferation.

    Journal: Oncotarget

    Article Title: Oncogenic transgelin-2 is differentially regulated in isocitrate dehydrogenase wild-type vs. mutant gliomas

    doi: 10.18632/oncotarget.26365

    Figure Lengend Snippet: (A) GBM30 neurospheres stably expressing TAGLN2 shRNA and corresponding scrambled shRNA control were generated and the level of stable TAGLN2 knock-down detected by Western blot is shown. (B) GBM30 neurospheres with stable knock-down of TAGLN2 or scrambled shRNA control were counted at 24, 72, and 1120 hours after plating. Knock-down of TAGLN2 resulted in significantly decreased cell counts (p<0.05). (C) GBM30 neurospheres and (D) U87 MG glioma cells stably overexpressing TAGLN2 and corresponding vector control were generated and the level of stable TAGLN2 overexpression was detected by Western blot. Of note, endogenous TAGLN2 (22 Kda) and exogenous TAGLN2 -myc (28 kDa) are shown. (E) GBM30 neurospheres stably overexpressing TAGLN2 resulted in significantly increased cell proliferation compared to vector control at 72 and 120 hours (p<0.05). (F) U87 MG cells stably overexpressing TAGLN2 resulted in increased cell proliferation compared to the vector alone. Experiments were performed twice with six replicates each. * , statistically significant difference in proliferation.

    Article Snippet: Since TAGLN2 was found to be expressed at significantly lower mRNA and protein levels in IDH1/2 mutant gliomas from both institutional and TCGA patient cohorts, we confirmed these findings in vitro by evaluating TAGLN2 protein levels in a commercially available U87 MG isogenic cell line overexpressing IDH1 R132H mutant protein (ATCC, Manassas, VA), which will be referred to as IDH1 mutant U87 MG cells.

    Techniques: Stable Transfection, Expressing, shRNA, Generated, Western Blot, Plasmid Preparation, Over Expression

    Since TAGLN2 has been shown to play a role in invasion and metastases of other cancer types, the invasive ability of (A) GBM30 neurospheres with stable shRNA-mediated knock-down of TAGLN2 were compared to their respective scrambled shRNA control. GBM30 cells showed a decrease in average number of cells invading through the matrix after knock-down of TAGLN2 compared to control. In contrast, (B) GBM30 neurospheres and (C) U87 MG glioma cells with stable overexpression of TAGLN2 showed an increase in average number of cells invading through the matrix compared to vector control. Experiments were performed three times with triplicate invasion assays. * , statistically significant difference in invading cells (p<0.05). Photographs are representative images at 40x and 100x magnification.

    Journal: Oncotarget

    Article Title: Oncogenic transgelin-2 is differentially regulated in isocitrate dehydrogenase wild-type vs. mutant gliomas

    doi: 10.18632/oncotarget.26365

    Figure Lengend Snippet: Since TAGLN2 has been shown to play a role in invasion and metastases of other cancer types, the invasive ability of (A) GBM30 neurospheres with stable shRNA-mediated knock-down of TAGLN2 were compared to their respective scrambled shRNA control. GBM30 cells showed a decrease in average number of cells invading through the matrix after knock-down of TAGLN2 compared to control. In contrast, (B) GBM30 neurospheres and (C) U87 MG glioma cells with stable overexpression of TAGLN2 showed an increase in average number of cells invading through the matrix compared to vector control. Experiments were performed three times with triplicate invasion assays. * , statistically significant difference in invading cells (p<0.05). Photographs are representative images at 40x and 100x magnification.

    Article Snippet: Since TAGLN2 was found to be expressed at significantly lower mRNA and protein levels in IDH1/2 mutant gliomas from both institutional and TCGA patient cohorts, we confirmed these findings in vitro by evaluating TAGLN2 protein levels in a commercially available U87 MG isogenic cell line overexpressing IDH1 R132H mutant protein (ATCC, Manassas, VA), which will be referred to as IDH1 mutant U87 MG cells.

    Techniques: shRNA, Over Expression, Plasmid Preparation

    (A) TAGLN2 protein levels were compared in U87 MG IDH1/2 WT parental cells and a commercially available U87 MG isogenic cell line overexpressing IDH1 with a heterozygous R132H mutation by Western blot analysis. TAGLN2 protein was decreased in IDH1 mutant cells compared to IDH1/2 WT cells. (B) U87 MG IDH1 mutant cells were treated with increasing concentrations of 5-azacytidine (5-AZA) demethylating agent and TAGLN2 protein was evaluated by Western blot. 5-AZA resulted in increasing TAGLN2 protein levels expression in a dose-dependent manner.

    Journal: Oncotarget

    Article Title: Oncogenic transgelin-2 is differentially regulated in isocitrate dehydrogenase wild-type vs. mutant gliomas

    doi: 10.18632/oncotarget.26365

    Figure Lengend Snippet: (A) TAGLN2 protein levels were compared in U87 MG IDH1/2 WT parental cells and a commercially available U87 MG isogenic cell line overexpressing IDH1 with a heterozygous R132H mutation by Western blot analysis. TAGLN2 protein was decreased in IDH1 mutant cells compared to IDH1/2 WT cells. (B) U87 MG IDH1 mutant cells were treated with increasing concentrations of 5-azacytidine (5-AZA) demethylating agent and TAGLN2 protein was evaluated by Western blot. 5-AZA resulted in increasing TAGLN2 protein levels expression in a dose-dependent manner.

    Article Snippet: Since TAGLN2 was found to be expressed at significantly lower mRNA and protein levels in IDH1/2 mutant gliomas from both institutional and TCGA patient cohorts, we confirmed these findings in vitro by evaluating TAGLN2 protein levels in a commercially available U87 MG isogenic cell line overexpressing IDH1 R132H mutant protein (ATCC, Manassas, VA), which will be referred to as IDH1 mutant U87 MG cells.

    Techniques: Mutagenesis, Western Blot, Expressing