Review

atm inhibitor ku60019 (MedChemExpress)

MedChemExpress is a verified supplier

MedChemExpress manufactures this product

About

News

Press Release

Team

Advisors

Partners

Contact

Bioz Stars

Bioz vStars

Buy from Supplier

Structured Review

MedChemExpress atm inhibitor ku60019

Oxidized ataxia telangiectasia mutated promotes triple-negative breast cancer-cancer stem cells enrichment and stemness maintenance. A: Representative images showing inhibition of mammosphere formation by the ataxia telangiectasia mutated inhibitor <t>Ku60019;</t> B: Quantification of mammosphere size; C: Quantification of mammosphere number; D-F: Effects of ataxia telangiectasia mutated knockdown on cancer stem cell enrichment (D), mammosphere size (E), and mammosphere number (F); G and H: Western blot analysis of stemness-associated proteins (c-Myc, sex-determining region Y-box 2, Kruppel-like factor 4, octamer-binding protein 4) following Ku60019 treatment (G) or ataxia telangiectasia mutated knockdown (H). Scale bar: 200 μm. Data were presented as mean ± SD ( n = 3). a P < 0.05; b P < 0.01. KLF4: Kruppel-like factor 4; SOX2: Sex-determining region Y-box 2; p-ATM: Phosphorylated ataxia telangiectasia mutated; shATM: Ataxia telangiectasia mutated knockdown.

Atm Inhibitor Ku60019, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 94/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/atm inhibitor ku60019/product/MedChemExpress
Average 94 stars, based on 31 article reviews

atm inhibitor ku60019 - by Bioz Stars, 2026-06

94/100 stars

Images

1) Product Images from "Hypoxia facilitates triple-negative breast cancer stem cells enrichment and stemness maintenance through oxidized ataxia telangiectasia mutated-induced one-carbon metabolism"

Article Title: Hypoxia facilitates triple-negative breast cancer stem cells enrichment and stemness maintenance through oxidized ataxia telangiectasia mutated-induced one-carbon metabolism

Journal: World Journal of Stem Cells

doi: 10.4252/wjsc.v18.i1.112278

Figure Legend Snippet: Oxidized ataxia telangiectasia mutated promotes triple-negative breast cancer-cancer stem cells enrichment and stemness maintenance. A: Representative images showing inhibition of mammosphere formation by the ataxia telangiectasia mutated inhibitor Ku60019; B: Quantification of mammosphere size; C: Quantification of mammosphere number; D-F: Effects of ataxia telangiectasia mutated knockdown on cancer stem cell enrichment (D), mammosphere size (E), and mammosphere number (F); G and H: Western blot analysis of stemness-associated proteins (c-Myc, sex-determining region Y-box 2, Kruppel-like factor 4, octamer-binding protein 4) following Ku60019 treatment (G) or ataxia telangiectasia mutated knockdown (H). Scale bar: 200 μm. Data were presented as mean ± SD ( n = 3). a P < 0.05; b P < 0.01. KLF4: Kruppel-like factor 4; SOX2: Sex-determining region Y-box 2; p-ATM: Phosphorylated ataxia telangiectasia mutated; shATM: Ataxia telangiectasia mutated knockdown.

Techniques Used: Inhibition, Knockdown, Western Blot, Binding Assay

Hypoxia and oxidized ataxia telangiectasia mutated facilitate metabolic remodeling in triple-negative breast cancer-cancer stem cells. A: Volcano plot showing differentially regulated metabolites (115 upregulated, 129 downregulated) in MDA-MB-231 cancer stem cells under hypoxia compared with normoxia; B: Pathway enrichment analysis of differentially regulated metabolites; C: Kyoto Encyclopedia of Genes and Genomes classification of altered metabolic pathways; D: Volcano plot showing metabolic changes in hypoxic cancer stem cells after Ku60019 treatment; E: Pathway enrichment analysis after Ku60019 treatment; F: Kyoto Encyclopedia of Genes and Genomes classification of altered metabolic pathways after Ku60019 treatment.

Figure Legend Snippet: Hypoxia and oxidized ataxia telangiectasia mutated facilitate metabolic remodeling in triple-negative breast cancer-cancer stem cells. A: Volcano plot showing differentially regulated metabolites (115 upregulated, 129 downregulated) in MDA-MB-231 cancer stem cells under hypoxia compared with normoxia; B: Pathway enrichment analysis of differentially regulated metabolites; C: Kyoto Encyclopedia of Genes and Genomes classification of altered metabolic pathways; D: Volcano plot showing metabolic changes in hypoxic cancer stem cells after Ku60019 treatment; E: Pathway enrichment analysis after Ku60019 treatment; F: Kyoto Encyclopedia of Genes and Genomes classification of altered metabolic pathways after Ku60019 treatment.

Techniques Used:

Oxidized ataxia telangiectasia mutated promotes serine hydroxymethyltransferase 2 and methylenetetrahydrofolate dehydrogenase 2 expression through c-Myc. A and B: Western blot analysis of phosphorylated ataxia telangiectasia mutated, c-Myc, serine hydroxymethyltransferase 2 (SHMT2), and methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) in Hs578T and MDA-MB-231 cells after treatment with Ku60019 and ataxia telangiectasia mutated knockdown; C: Consensus c-Myc binding motif; D: Schematic representation of predicted c-Myc binding sites in the human MTHFD2 and SHMT2 promoter regions; E: Luciferase assay showed SHMT2 and MTHFD2 relative luciferase activity; F: Representative chromatin immunoprecipitation (ChIP)-polymerase chain reaction (PCR) showing c-Myc occupancy at the MTHFD2 and SHMT2 promoters; input and immunoglobulin G served as controls; G and H: ChIP-quantitative PCR analysis demonstrating c-Myc enrichment at the MTHFD2 (G) and SHMT2 (H) promoters in Hs578T and MDA-MB-231 cells. ChIP-quantitative PCR enrichment expressed as % input relative to immunoglobulin G. Data were presented as mean ± SD ( n = 3). a P < 0.05; b P < 0.01. p-ATM: Phosphorylated ataxia telangiectasia mutated; MTHFD2: Methylenetetrahydrofolate dehydrogenase 2; SHMT2: Serine hydroxymethyltransferase 2; IgG: Immunoglobulin G.

Figure Legend Snippet: Oxidized ataxia telangiectasia mutated promotes serine hydroxymethyltransferase 2 and methylenetetrahydrofolate dehydrogenase 2 expression through c-Myc. A and B: Western blot analysis of phosphorylated ataxia telangiectasia mutated, c-Myc, serine hydroxymethyltransferase 2 (SHMT2), and methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) in Hs578T and MDA-MB-231 cells after treatment with Ku60019 and ataxia telangiectasia mutated knockdown; C: Consensus c-Myc binding motif; D: Schematic representation of predicted c-Myc binding sites in the human MTHFD2 and SHMT2 promoter regions; E: Luciferase assay showed SHMT2 and MTHFD2 relative luciferase activity; F: Representative chromatin immunoprecipitation (ChIP)-polymerase chain reaction (PCR) showing c-Myc occupancy at the MTHFD2 and SHMT2 promoters; input and immunoglobulin G served as controls; G and H: ChIP-quantitative PCR analysis demonstrating c-Myc enrichment at the MTHFD2 (G) and SHMT2 (H) promoters in Hs578T and MDA-MB-231 cells. ChIP-quantitative PCR enrichment expressed as % input relative to immunoglobulin G. Data were presented as mean ± SD ( n = 3). a P < 0.05; b P < 0.01. p-ATM: Phosphorylated ataxia telangiectasia mutated; MTHFD2: Methylenetetrahydrofolate dehydrogenase 2; SHMT2: Serine hydroxymethyltransferase 2; IgG: Immunoglobulin G.

Techniques Used: Expressing, Western Blot, Knockdown, Binding Assay, Luciferase, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

Oxidized ataxia telangiectasia mutated sustains triple-negative breast cancer-cancer stem cells stemness via serine hydroxymethyltransferase 2- and methylenetetrahydrofolate dehydrogenase 2-mediated one-carbon metabolism. A: NADPH/NADP+ ratio in cancer stem cells (CSCs) under hypoxia compared with normoxia; B and C: NADPH/NADP+ ratio decreased in CSCs after Ku60019 treatment (B) or shc-Myc knockdown (C); D-F: Effects of one-carbon metabolite supplementation on mammosphere size (D) and number (E and F) in CSCs with serine hydroxymethyltransferase 2 (SHMT2) knockdown or methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) knockdown; G: Western blot analysis of MTHFD2, SHMT2, Kruppel-like factor 4, and sex-determining region Y-box 2 expression following SHMT2 or MTHFD2 knockdown under hypoxia. Data were presented as mean ± SD ( n = 3). a P < 0.05. MTHFD2: Methylenetetrahydrofolate dehydrogenase 2; SHMT2: Serine hydroxymethyltransferase 2; KLF4: Kruppel-like factor 4; SOX2: Sex-determining region Y-box 2.

Figure Legend Snippet: Oxidized ataxia telangiectasia mutated sustains triple-negative breast cancer-cancer stem cells stemness via serine hydroxymethyltransferase 2- and methylenetetrahydrofolate dehydrogenase 2-mediated one-carbon metabolism. A: NADPH/NADP+ ratio in cancer stem cells (CSCs) under hypoxia compared with normoxia; B and C: NADPH/NADP+ ratio decreased in CSCs after Ku60019 treatment (B) or shc-Myc knockdown (C); D-F: Effects of one-carbon metabolite supplementation on mammosphere size (D) and number (E and F) in CSCs with serine hydroxymethyltransferase 2 (SHMT2) knockdown or methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) knockdown; G: Western blot analysis of MTHFD2, SHMT2, Kruppel-like factor 4, and sex-determining region Y-box 2 expression following SHMT2 or MTHFD2 knockdown under hypoxia. Data were presented as mean ± SD ( n = 3). a P < 0.05. MTHFD2: Methylenetetrahydrofolate dehydrogenase 2; SHMT2: Serine hydroxymethyltransferase 2; KLF4: Kruppel-like factor 4; SOX2: Sex-determining region Y-box 2.

Techniques Used: Knockdown, Western Blot, Expressing

Image Search Results

Journal: World Journal of Stem Cells

Article Title: Hypoxia facilitates triple-negative breast cancer stem cells enrichment and stemness maintenance through oxidized ataxia telangiectasia mutated-induced one-carbon metabolism

doi: 10.4252/wjsc.v18.i1.112278

Figure Lengend Snippet: Oxidized ataxia telangiectasia mutated promotes triple-negative breast cancer-cancer stem cells enrichment and stemness maintenance. A: Representative images showing inhibition of mammosphere formation by the ataxia telangiectasia mutated inhibitor Ku60019; B: Quantification of mammosphere size; C: Quantification of mammosphere number; D-F: Effects of ataxia telangiectasia mutated knockdown on cancer stem cell enrichment (D), mammosphere size (E), and mammosphere number (F); G and H: Western blot analysis of stemness-associated proteins (c-Myc, sex-determining region Y-box 2, Kruppel-like factor 4, octamer-binding protein 4) following Ku60019 treatment (G) or ataxia telangiectasia mutated knockdown (H). Scale bar: 200 μm. Data were presented as mean ± SD ( n = 3). a P < 0.05; b P < 0.01. KLF4: Kruppel-like factor 4; SOX2: Sex-determining region Y-box 2; p-ATM: Phosphorylated ataxia telangiectasia mutated; shATM: Ataxia telangiectasia mutated knockdown.

Article Snippet: The ATM inhibitor Ku60019 was purchased from MedChem Express.

Techniques: Inhibition, Knockdown, Western Blot, Binding Assay

Journal: World Journal of Stem Cells

Article Title: Hypoxia facilitates triple-negative breast cancer stem cells enrichment and stemness maintenance through oxidized ataxia telangiectasia mutated-induced one-carbon metabolism

doi: 10.4252/wjsc.v18.i1.112278

Figure Lengend Snippet: Hypoxia and oxidized ataxia telangiectasia mutated facilitate metabolic remodeling in triple-negative breast cancer-cancer stem cells. A: Volcano plot showing differentially regulated metabolites (115 upregulated, 129 downregulated) in MDA-MB-231 cancer stem cells under hypoxia compared with normoxia; B: Pathway enrichment analysis of differentially regulated metabolites; C: Kyoto Encyclopedia of Genes and Genomes classification of altered metabolic pathways; D: Volcano plot showing metabolic changes in hypoxic cancer stem cells after Ku60019 treatment; E: Pathway enrichment analysis after Ku60019 treatment; F: Kyoto Encyclopedia of Genes and Genomes classification of altered metabolic pathways after Ku60019 treatment.

Article Snippet: The ATM inhibitor Ku60019 was purchased from MedChem Express.

Techniques:

Journal: World Journal of Stem Cells

Article Title: Hypoxia facilitates triple-negative breast cancer stem cells enrichment and stemness maintenance through oxidized ataxia telangiectasia mutated-induced one-carbon metabolism

doi: 10.4252/wjsc.v18.i1.112278

Figure Lengend Snippet: Oxidized ataxia telangiectasia mutated promotes serine hydroxymethyltransferase 2 and methylenetetrahydrofolate dehydrogenase 2 expression through c-Myc. A and B: Western blot analysis of phosphorylated ataxia telangiectasia mutated, c-Myc, serine hydroxymethyltransferase 2 (SHMT2), and methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) in Hs578T and MDA-MB-231 cells after treatment with Ku60019 and ataxia telangiectasia mutated knockdown; C: Consensus c-Myc binding motif; D: Schematic representation of predicted c-Myc binding sites in the human MTHFD2 and SHMT2 promoter regions; E: Luciferase assay showed SHMT2 and MTHFD2 relative luciferase activity; F: Representative chromatin immunoprecipitation (ChIP)-polymerase chain reaction (PCR) showing c-Myc occupancy at the MTHFD2 and SHMT2 promoters; input and immunoglobulin G served as controls; G and H: ChIP-quantitative PCR analysis demonstrating c-Myc enrichment at the MTHFD2 (G) and SHMT2 (H) promoters in Hs578T and MDA-MB-231 cells. ChIP-quantitative PCR enrichment expressed as % input relative to immunoglobulin G. Data were presented as mean ± SD ( n = 3). a P < 0.05; b P < 0.01. p-ATM: Phosphorylated ataxia telangiectasia mutated; MTHFD2: Methylenetetrahydrofolate dehydrogenase 2; SHMT2: Serine hydroxymethyltransferase 2; IgG: Immunoglobulin G.

Article Snippet: The ATM inhibitor Ku60019 was purchased from MedChem Express.

Techniques: Expressing, Western Blot, Knockdown, Binding Assay, Luciferase, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

Journal: World Journal of Stem Cells

Article Title: Hypoxia facilitates triple-negative breast cancer stem cells enrichment and stemness maintenance through oxidized ataxia telangiectasia mutated-induced one-carbon metabolism

doi: 10.4252/wjsc.v18.i1.112278

Figure Lengend Snippet: Oxidized ataxia telangiectasia mutated sustains triple-negative breast cancer-cancer stem cells stemness via serine hydroxymethyltransferase 2- and methylenetetrahydrofolate dehydrogenase 2-mediated one-carbon metabolism. A: NADPH/NADP+ ratio in cancer stem cells (CSCs) under hypoxia compared with normoxia; B and C: NADPH/NADP+ ratio decreased in CSCs after Ku60019 treatment (B) or shc-Myc knockdown (C); D-F: Effects of one-carbon metabolite supplementation on mammosphere size (D) and number (E and F) in CSCs with serine hydroxymethyltransferase 2 (SHMT2) knockdown or methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) knockdown; G: Western blot analysis of MTHFD2, SHMT2, Kruppel-like factor 4, and sex-determining region Y-box 2 expression following SHMT2 or MTHFD2 knockdown under hypoxia. Data were presented as mean ± SD ( n = 3). a P < 0.05. MTHFD2: Methylenetetrahydrofolate dehydrogenase 2; SHMT2: Serine hydroxymethyltransferase 2; KLF4: Kruppel-like factor 4; SOX2: Sex-determining region Y-box 2.

Article Snippet: The ATM inhibitor Ku60019 was purchased from MedChem Express.

Techniques: Knockdown, Western Blot, Expressing

Effects of ATM inhibition on cisplatin-treated A549 cell viability. Cell viability was measured via MTT assay after 72 h of treatment and normalized to baseline viability before treatment. DMSO—vehicle control (0.2%), SP—SP600125 (20 μM), KU—KU60019 (5 μM), and combo—SP + KU. Statistical significance is indicated by asterisks (n.s.—non-significant; ***— p < 0.0005; more details in the Methods and ).

Journal: Pharmaceuticals

Article Title: Potential Mechanisms of MAP Kinase JNK’s Involvement in Modulating Cancer Cell Fate in a Cisplatin Concentration-Dependent Manner

doi: 10.3390/ph19030509

Figure Lengend Snippet: Effects of ATM inhibition on cisplatin-treated A549 cell viability. Cell viability was measured via MTT assay after 72 h of treatment and normalized to baseline viability before treatment. DMSO—vehicle control (0.2%), SP—SP600125 (20 μM), KU—KU60019 (5 μM), and combo—SP + KU. Statistical significance is indicated by asterisks (n.s.—non-significant; ***— p < 0.0005; more details in the Methods and ).

Article Snippet: Other reagents: 99.5% dimethylsulphoxide DMSO (#7029.1) from Carl Roth, Karlsruhe, Germany; oxaliplatin (5 mg/mL infusion solution) from Fresenius Kabi Oncology Plc., Hampshire, UK; daunorubicin (#251800) and AKT inhibitor VIII (10 μM, #124018) from EMD Millipore/Merck, Billerica, MA, USA; ATM inhibitor KU60019 (5 μM, #S1570) and RAS inhibitor RMC-6236 (1 μM, #E1597) from Selleck Chemicals LLC, Houston, TX, USA; SP600125 (20 μM; #J67272) from Alpha Aesar, Ward Hill, MA, USA; capivasertib AZD5363 (10 μM; #15406) from Cayman Chemical, Ann Arbor, MI, USA.

Techniques: Inhibition, MTT Assay, Control

Induction of H2AX phosphorylation by cisplatin. ( a ) High concentration of cisplatin induces H2AX phosphorylation as early as 6 h after the treatment. ( b ) After 20 h, even very low concentrations of cisplatin induce H2AX phosphorylation. ( c ) ATM inhibitor reduces H2AX phosphorylation. DM—vehicle control DMSO (0.2%) and KU—KU60019 (5 μM). Representative Western blots are shown. Total protein stained with Coomassie dye in polyacrylamide gels serve as loading controls.

Journal: Pharmaceuticals

Article Title: Potential Mechanisms of MAP Kinase JNK’s Involvement in Modulating Cancer Cell Fate in a Cisplatin Concentration-Dependent Manner

doi: 10.3390/ph19030509

Figure Lengend Snippet: Induction of H2AX phosphorylation by cisplatin. ( a ) High concentration of cisplatin induces H2AX phosphorylation as early as 6 h after the treatment. ( b ) After 20 h, even very low concentrations of cisplatin induce H2AX phosphorylation. ( c ) ATM inhibitor reduces H2AX phosphorylation. DM—vehicle control DMSO (0.2%) and KU—KU60019 (5 μM). Representative Western blots are shown. Total protein stained with Coomassie dye in polyacrylamide gels serve as loading controls.

Techniques: Phospho-proteomics, Concentration Assay, Control, Western Blot, Staining

Effect of ATM kinase inhibition on p53 expression. ( a ) In A549 cells treated with 25 μM cisplatin (both alone and in combination with the JNK inhibitor SP600125), p53 expression is decreased after the addition of the ATM inhibitor KU60019 (KU, 5 μM). ( b ) In A549 cells exposed to 100 μM cisplatin and JNK inhibitor SP600125, ATM inhibitor KU decreases p53 expression. DM—vehicle control DMSO (0.2%), SP—SP600125 (20 μM); treatment duration—20 h. Representative Western blots are shown. Total protein stained with Coomassie dye in polyacrylamide gels serve as loading controls.

Journal: Pharmaceuticals

Article Title: Potential Mechanisms of MAP Kinase JNK’s Involvement in Modulating Cancer Cell Fate in a Cisplatin Concentration-Dependent Manner

doi: 10.3390/ph19030509

Figure Lengend Snippet: Effect of ATM kinase inhibition on p53 expression. ( a ) In A549 cells treated with 25 μM cisplatin (both alone and in combination with the JNK inhibitor SP600125), p53 expression is decreased after the addition of the ATM inhibitor KU60019 (KU, 5 μM). ( b ) In A549 cells exposed to 100 μM cisplatin and JNK inhibitor SP600125, ATM inhibitor KU decreases p53 expression. DM—vehicle control DMSO (0.2%), SP—SP600125 (20 μM); treatment duration—20 h. Representative Western blots are shown. Total protein stained with Coomassie dye in polyacrylamide gels serve as loading controls.

Techniques: Inhibition, Expressing, Control, Western Blot, Staining

Effect of ATM inhibition on protein kinase AKT phosphorylation. ( a ) In A549 cells treated with 25 μM cisplatin (both alone and in combination with the JNK inhibitor SP600125), AKT phosphorylation is decreased after the addition of the ATM inhibitor KU60019 (KU, 5 μM). ( b ) In A549 cells exposed to 100 μM cisplatin and JNK inhibitor SP600125, ATM inhibitor KU decreases AKT phosphorylation. DM—vehicle control DMSO (0.2%) and SP—SP600125 (20 μM); treatment duration—20 h. Representative Western blots are shown. Total protein stained with Coomassie dye in polyacrylamide gels serve as loading controls.

Journal: Pharmaceuticals

Article Title: Potential Mechanisms of MAP Kinase JNK’s Involvement in Modulating Cancer Cell Fate in a Cisplatin Concentration-Dependent Manner

doi: 10.3390/ph19030509

Figure Lengend Snippet: Effect of ATM inhibition on protein kinase AKT phosphorylation. ( a ) In A549 cells treated with 25 μM cisplatin (both alone and in combination with the JNK inhibitor SP600125), AKT phosphorylation is decreased after the addition of the ATM inhibitor KU60019 (KU, 5 μM). ( b ) In A549 cells exposed to 100 μM cisplatin and JNK inhibitor SP600125, ATM inhibitor KU decreases AKT phosphorylation. DM—vehicle control DMSO (0.2%) and SP—SP600125 (20 μM); treatment duration—20 h. Representative Western blots are shown. Total protein stained with Coomassie dye in polyacrylamide gels serve as loading controls.

Techniques: Inhibition, Phospho-proteomics, Control, Western Blot, Staining

(A) Immunoblots with lysates from U2OS cells transiently expressing FLAG-tagged RHEB, pre-treated with ATM inhibitor (ATMi; KU-60019, 1 μM, 30 min), followed by treatment with etoposide (ETO; 20 μM, 2 hr), probed with the indicated antibodies. DMSO was used as control for all treatments. n = 3 independent experiments. (B) Immunoblots with lysates from RHEB KO HEK293FT cells stably expressing FLAG-tagged RHEB, pre-treated with ATM inhibitor (ATMi; KU-60019; 1 μM), ATR inhibitor (ATRi; VE-832; 1 μM), or DNA-PK inhibitor (DNA-PKi; NU7441; 0.1 μM or 1 μM) for 30 min, followed by treatment with etoposide (ETO; 20 μM, 2 hr), probed with the indicated antibodies. DMSO was used as control for all treatments. n = 3 independent experiments. (C) Immunoblots with lysates from RHEB KO HEK293FT cells stably expressing FLAG-tagged RHEB, transfected with siRNAs targeting ATM (siATM), ATR (siATR), or Luciferase as a control, probed with the indicated antibodies. Cells were treated with etoposide (ETO; 20 μM, 2 hr), or DMSO as control, prior to lysis.

Journal: bioRxiv

Article Title: ATM-dependent RHEB phosphorylation couples DNA damage to lysosomal mTORC1 signaling to orchestrate the cellular response to genotoxic stress

doi: 10.64898/2026.01.16.699946

Figure Lengend Snippet: (A) Immunoblots with lysates from U2OS cells transiently expressing FLAG-tagged RHEB, pre-treated with ATM inhibitor (ATMi; KU-60019, 1 μM, 30 min), followed by treatment with etoposide (ETO; 20 μM, 2 hr), probed with the indicated antibodies. DMSO was used as control for all treatments. n = 3 independent experiments. (B) Immunoblots with lysates from RHEB KO HEK293FT cells stably expressing FLAG-tagged RHEB, pre-treated with ATM inhibitor (ATMi; KU-60019; 1 μM), ATR inhibitor (ATRi; VE-832; 1 μM), or DNA-PK inhibitor (DNA-PKi; NU7441; 0.1 μM or 1 μM) for 30 min, followed by treatment with etoposide (ETO; 20 μM, 2 hr), probed with the indicated antibodies. DMSO was used as control for all treatments. n = 3 independent experiments. (C) Immunoblots with lysates from RHEB KO HEK293FT cells stably expressing FLAG-tagged RHEB, transfected with siRNAs targeting ATM (siATM), ATR (siATR), or Luciferase as a control, probed with the indicated antibodies. Cells were treated with etoposide (ETO; 20 μM, 2 hr), or DMSO as control, prior to lysis.

Article Snippet: For kinase activity inhibition, cells were pre-treated with KU-60019 (ATM inhibitor; #S1570, Selleckchem; 1 μM) VE-821 (ATR inhibitor; #S8007, Selleckchem; 1 μM), and NU7441 (DNA-PK inhibitor; #S2638, Selleckchem; 0.1 or 1 μM) for 30 minutes prior to addition of DNA-damaging agents.

Techniques: Western Blot, Expressing, Control, Stable Transfection, Transfection, Luciferase, Lysis

(A) Immunoblots with lysates from RHEB KO HEK293FT cells stably expressing FLAG-tagged RHEB, transfected with siRNAs targeting TSC2 (siTSC2) or Luciferase as a control (siCtrl), probed with the indicated antibodies. Cells were treated with etoposide (ETO; 20 μM, 2 hr), or DMSO as control, prior to lysis. (B) Immunoblots with lysates from HEK293FT WT or TSC1 KO cells transiently expressing FLAG-tagged RHEB, treated with etoposide (ETO; 20 μM, 2 hr) or DMSO as control, probed with the indicated antibodies. (C) Co-immunoprecipitation between endogenous ATM and RHEB from HEK293FT cells transiently expressing FLAG-tagged RHEB or Luciferase (Luc) as a control. The input and IP samples were analyzed by immunoblotting with the indicated antibodies. (D) In vitro kinase assays with ATM immunopurified from HEK293FT cells transiently expressing FLAG-tagged WT or kinase-dead (KD) ATM, using recombinant His 6 -tagged RHEB protein as a substrate. Cells were treated with etoposide (ETO; 20 μM, 2 hr) before lysis to activate ATM. Specificity of RHEB phosphorylation was confirmed by pre-treatment with an ATM inhibitor (ATMi; KU-60019; 1 μM, 30 min), or omitting ATP from the reaction. Recombinant GST or GST-4E-BP1 proteins were used as a negative or positive control, respectively. Substrate phosphorylation detected by autoradiography. n = 3 independent experiments.

Journal: bioRxiv

Article Title: ATM-dependent RHEB phosphorylation couples DNA damage to lysosomal mTORC1 signaling to orchestrate the cellular response to genotoxic stress

doi: 10.64898/2026.01.16.699946

Figure Lengend Snippet: (A) Immunoblots with lysates from RHEB KO HEK293FT cells stably expressing FLAG-tagged RHEB, transfected with siRNAs targeting TSC2 (siTSC2) or Luciferase as a control (siCtrl), probed with the indicated antibodies. Cells were treated with etoposide (ETO; 20 μM, 2 hr), or DMSO as control, prior to lysis. (B) Immunoblots with lysates from HEK293FT WT or TSC1 KO cells transiently expressing FLAG-tagged RHEB, treated with etoposide (ETO; 20 μM, 2 hr) or DMSO as control, probed with the indicated antibodies. (C) Co-immunoprecipitation between endogenous ATM and RHEB from HEK293FT cells transiently expressing FLAG-tagged RHEB or Luciferase (Luc) as a control. The input and IP samples were analyzed by immunoblotting with the indicated antibodies. (D) In vitro kinase assays with ATM immunopurified from HEK293FT cells transiently expressing FLAG-tagged WT or kinase-dead (KD) ATM, using recombinant His 6 -tagged RHEB protein as a substrate. Cells were treated with etoposide (ETO; 20 μM, 2 hr) before lysis to activate ATM. Specificity of RHEB phosphorylation was confirmed by pre-treatment with an ATM inhibitor (ATMi; KU-60019; 1 μM, 30 min), or omitting ATP from the reaction. Recombinant GST or GST-4E-BP1 proteins were used as a negative or positive control, respectively. Substrate phosphorylation detected by autoradiography. n = 3 independent experiments.

Techniques: Western Blot, Stable Transfection, Expressing, Transfection, Luciferase, Control, Lysis, Immunoprecipitation, In Vitro, Recombinant, Phospho-proteomics, Positive Control, Autoradiography

Review

Structured Review

Images

1) Product Images from "Hypoxia facilitates triple-negative breast cancer stem cells enrichment and stemness maintenance through oxidized ataxia telangiectasia mutated-induced one-carbon metabolism"

Similar Products

Image Search Results