(a) Immunoblots showing protein levels of ATM, NBS1, and RAP80 in human skin fibroblasts (HSFs). A gradually increased level of p16 indicates cellular senescence, while elevated γH2AX level …
Statistical analysis for SA-β-Gal positive staining.
Statistical analysis for EdU positive staining.
(a) Real-time PCR analysis showing progressively elevated mRNA level of p21 in continuously cultured human endothelial cells (HUVEC). **p<0.01. (b) SA-β-Gal staining of HUVEC cells at indicated …
(a) Representative images showing cells treated with Scramble (sh-NC) or sh-ATM. (b) Percent EdU-positive cells in sh-NC or sh-ATM treated HSFs. Views were randomly captured and at least 100 cells …
(a) Quantitative RT-PCR analysis of mRNA levels of indicated glycolytic genes in different passages of MEFs with or without treatment of CQ. Data represent means ± SEM. *p<0.05, **p<0.01, ***p<0.001.…
Differently expressed mRNA profiles of Atm-/- MEF cells.
Differentially expressed Metabolites in Atm KO MEFs.
Differently expressed mRNA profiles of Sirt6-/- MEF cells.
(a) Quantitative RT-PCR analysis of mRNA levels of indicated glycolytic genes in different passages of HSFs with or without treatment of CQ. Data represent means ± SEM. *p<0.05, **p<0.01, ***p<0.001.…
(a) Heatmap representation of RNA-Seq data showing relative changes of glycolytic genes in Sirt6-/- MEF cells. Red and green indicate up- and downregulation, respectively. (b) Quantitative RT-PCR …
(a) Immunoblots showing protein levels of SIRT6 in HEK293 cells expressing Flag-ATM or Flag-ATM S1981A. (b) Immunoblots showing endogenous ATM and p-S/TQ motif in anti-Flag immunoprecipitates in …
(a) Immunoblots showing a significant increase in SIRT6 protein level, pS1981ATM, and pS824KAP-1 in the presence of DNA damage reagents-CPT and Dox in U2OS cells. (b) Immunoblots showing Sirt6 …
(a) Immunoblots showing protein levels of Flag-SIRT6 in WT and ATM KO HEK293 cells treated with CHX (50 µg/ml) for indicated periods of time. (b) Quantification of protein levels in (a) by ImageJ. …
(a) Immunoblots showing Sirt6 levels in wild-type and Atm null MEFs in the presence of CHX (50 μg/ml). (b–c) Immunoblots showing SIRT6 protein level in HEK293 cells in the presence of CHX (50 μg/ml) …
(a) Survival analysis of C. elegans treated with the indicated dosage of CQ. **p<0.01. NS indicates no significant difference. (b) Survival analysis of wild-type and atm-1 null C. elegans cultured …
Lifespan analysis of chloroquine's effect on nematodes.
Lifespan analysis of chloroquine's effect on mice.
(a) Survival analysis of sir-2.4 downregulated C. elegans exposed to medium with or without 1 µM CQ. ‘N.S.’ indicates no significant difference. (b) Quantitative RT-PCR analysis of mRNA levels of sir…
Left, DNA damage activates DDR cascade, and its constant activation leads to permanent cell cycle exit and senescence. Right, defective ATM-SIRT6 axis underlies premature aging in mouse models …
(a) MEFs at passage 1 were cultured in 20% O2 (black) or 3% O2 (grey) with treatment of 1 μM CQ (circles) or DMSO (squares), and cell number was determined at each passage. ***P < 0.01. (b) …
(a) The strategy for constructing ATM-1 overexpressing plasmid with GFP-tag. (b) Representative images showing the detectable GFP expression in worms. (c) Survival analysis of worm line expressing …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Gene (human) | ATM | National Center for Biotechnology Information | Gene ID: 472 | |
Gene (mouse) | Atm | National Center for Biotechnology Information | Gene ID: 11920 | |
Gene (Caenorhabditis elegans) | atm-1 | National Center for Biotechnology Information | Gene ID: 3565793 | |
Gene (human) | SIRT6 | National Center for Biotechnology Information | Gene ID: 51548 | |
Gene (mouse) | Sirt6 | National Center for Biotechnology Information | Gene ID: 50721 | |
Gene (Caenorhabditis elegans) | sir-2.4 | National Center for Biotechnology Information | Gene ID: 182284 | |
Gene (mouse) | Zmpste24 | National Center for Biotechnology Information | Gene ID: 230709 | |
Gene (mouse) | p53 | National Center for Biotechnology Information | Gene ID: 230710 | |
Cell line (human) | HEK293 | ATCC | Catalog number: ATCC CRL-1573; RRID:CVCL_0042 | |
Cell line (human) | HepG2 | ATCC | Catalog number: ATCC HB-8065; RRID:CVCL_0027 | |
Cell line (human) | U2OS | ATCC | Catalog number: ATCC HTB-96; RRID:CVCL_0042 | |
Cell line (mouse) | Atm-/-; p53-/- MEF | from Dr. Yosef Shiloh (Tel Aviv University, Israel) | ||
Cell line (mouse) | Sirt6-/- MEF | from Dr. Raul Mostoslavsky (Massachusetts General Hospital Cancer center, USA) | ||
Antibody | ATM | Abcam (Cambridge, UK) | Cat# ab78; RRID:AB_306089 | Applications: WB;Immunofluorescence |
Antibody | SIRT6 | Abcam (Cambridge, UK) | Cat# ab62739; RRID:AB_956300 | Applications: WB; Immunofluoresce;Chromatin immunoprecipitation |
Antibody | γH2AX | Abcam (Cambridge, UK) | Cat# ab81299; RRID:AB_1640564 | Applications: WB |
Antibody | p21 | Santa Cruz Biotechnology | Cat# sc-6246; RRID:AB_628073 | Applications: WB |
Antibody | MDM2 | Santa Cruz Biotechnology | Cat# sc-965; RRID:AB_627920 | Applications: WB |
Antibody | p-ATM (Ser1981) | EMD Millipore | Cat# 05–740; RRID:AB_309954 | Applications: WB |
Antibody | H3K9ac | EMD Millipore | Cat# 07–352; RRID:AB_310544 | Applications: WB; Chromatin immunoprecipitation |
Antibody | p-S/T Q | Cell Signaling Technology (Beverly, MA) | Cat #9607S; RRID:AB_10889739 | Applications: WB |
Antibody | cleaved caspase-3 | Cell Signaling Technology (Beverly, MA) | Cat #9661; RRID:AB_2341188 | Applications: WB |
Antibody | HA | Sigma-Aldrich | Cat# H3663; RRID:AB_262051 | Applications: WB |
Antibody | Flag | Sigma-Aldrich | Cat# F1804; RRID: AB_262044 | Applications: WB |
Antibody | LC3B | Sigma-Aldrich | Cat# L7543; RRID:AB_796155 | Applications: WB |
Transfected construct (human) | Flag-His-ATM wt | Addgene (Cambridge, MA) | Cat #31985 | |
Transfected construct (human) | Flag-SIRT6 | Addgene (Cambridge, MA) | Cat #13817 | |
Transfected construct (human) | Flag-His-ATM S1981A | Addgene (Cambridge, MA) | Cat #32300 | |
Commercial assay or kit | Senescence beta- galactosidase staining Kit | Cell Signaling Technology (Beverly, MA) | Cat #9860 | |
Commercial assay or kit | Lactate Colorimetric Assay Kit | BioVision | Cat #K667-100 | |
Commercial assay or kit | Click-iT EdU Alexa Fluor 488 Kit | Invitrogen | Cat #C10425 | |
Chemical compound, drug | Cycloheximide | Sigma-Aldrich | Cat #66-81-9 | |
Chemical compound, drug | MG-132 | Sigma-Aldrich | Cat #474787 | |
Chemical compound, drug | Chloroquine | Sigma-Aldrich | Cat #C6628 |