Journal: Cell Death Discovery

Article Title: The role of declining ataxia-telangiectasia-mutated (ATM) function in oocyte aging

doi: 10.1038/s41420-024-02041-z

Figure Lengend Snippet: Confirmation of the efficacy of RNA interference with ATM siRNA in mouse oocytes by microinjection. ATM siRNA injection effectively reduced ATM gene and protein expression in mouse oocytes. The experiments were replicated a minimum of five times. A The bar graph shows a significantly lower relative ATM expression in the ATM knockdown (ATM-KD) group, compared to Uninjected (UI) and Scrambled (SCR) controls (oocytes from 6–8 weeks old mice). **** p < 0.0001, Kruskal–Wallis test. n = 38 in ATM-KD group, n = 27 in SCR, n = 38 in UI. Results in mean ± SEM. A.U. (arbitrary units). B The bar graph shows a significantly lower ATM fluorescence intensity in the ATM-KD group, compared with the UI and SCR controls. *** p < 0.0005, One-way ANOVA test. n = 14 in ATM-KD group, n = 18 in SCR, n = 35 in UI. Results in mean ± SEM. AU arbitrary units. C Representative confocal laser scanning microscope images illustrate lower ATM protein expression in mouse oocytes after ATM knockdown. Cytoplasmic ATM protein expression is shown in green, and the oocytes were counterstained with DAPI in blue.

Article Snippet: After removing cumulus cells by pipetting, the retrieved oocytes were microinjected with either 50 μM of scrambled (SCR) (REF: sc-36869, Santa Cruz Biotechnology, Inc., Texas, USA) or ATM siRNA (REF: sc-29762, Santa Cruz Biotechnology, Inc.) in modified HTF medium (REF: 90126, FUJIFILM, Japan) supplemented with 0.1 mM 3-isobutyl-1-methylxanthine (IBMX) (REF: I5879, Sigma-Aldrich, Missouri, United States).

Techniques: Microinjection, Injection, Expressing, Knockdown, Fluorescence, Laser-Scanning Microscopy

Journal: Cancer & Metabolism

Article Title: ATM inhibition blocks glucose metabolism and amplifies the sensitivity of resistant lung cancer cell lines to oncogene driver inhibitors

doi: 10.1186/s40170-023-00320-4

Figure Lengend Snippet: Effects of ATM silencing in oncogene-driven H1993 and H1975 cells. A Levels of HKII, phospho-PKM2 Tyr105 , PKM2, phospho-PDH Ser293 , PDH, cyclin D1, and BIM in H1993 and H1975 cells transfected with ATM-targeted small interfering RNA or siRNA control and exposed to crizotinib or WZ4002 for 48 h. B Levels of OXPHOS in H1993 and H1975 cells transfected with ATM-targeted small interfering RNA or siRNA control and exposed to crizotinib or WZ4002 for 48 h. GAPDH and actin serve as equal loading controls

Article Snippet: H1993 and H1975 cells were transfected with siRNA targeting ATM (sense CUUAGCAGGAGGUGUAAAU, antisense AUUUACACCUCCUGCUAAG) and control non-targeting siRNA (siCTRL) purchased from Sigma-Aldrich and used according to the manufacturer’s instructions.

Techniques: Transfection, Small Interfering RNA

Journal: PLoS ONE

Article Title: Alterations in Cellular Energy Metabolism Associated with the Antiproliferative Effects of the ATM Inhibitor KU-55933 and with Metformin

doi: 10.1371/journal.pone.0049513

Figure Lengend Snippet: ( A ) MCF-7 (LKB +/+ ) and ( B ) HeLa (LKB −/− ) cancer cells in exponential stages of growth were seeded into 96-well plates with 10% FBS and after 24 hrs exposed to increasing concentrations of KU-55933 (ATM inhibitor) in media containing 1% FBS for 72 hrs. Cell growth was estimated by Alamar Blue dye reduction (resazurin (3 µM)). Data are presented as mean ± S.E.M. from 3 independent experiments done in triplicate. ( C–F ) MCF-7 HepG2, HeLa and MCF-10A cells were growth inhibited by KU-55933 and metformin. Cells were seeded into 96-well plates in the presence of 1% FBS and after 24 hrs treated with KU-55933 (10 µM) or metformin (5 mM). Data are presented as mean ± S.E.M. from 4 independent experiments done in triplicate. * indicates a result significantly different from that obtained in the absence of KU-55933 or metformin as determined by 2-way ANOVA ( P <0.0001). ( G ) MCF-7 cells were transfected with 50 nM ATM-siRNA or with control siRNA. Twenty-four hours after transfection, cells were treated with KU-55933 (10 µM) or metformin (5 mM) and incubated for 48 hrs in RPMI containing 1% FBS. Cell growth in each well was measured by counting cells using Trypan blue. Results using cell number or Alamar blue as endpoints yielded the same conclusions. Columns, mean of 3 independent experiments carried out in triplicate (n = 9); bars, S.E.M. ( H ) After transfecting MCF-7 cells with 50 nM ATM-siRNA or with control siRNA, cells were lysed and prepared for immunoblot analyses using antibodies against ATM. ß-actin is shown as a loading control.

Article Snippet: Metformin (1, 1-Dimethylbiguanide hydrchloride), rotenone and FCCP, (Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone) were purchased from Sigma-Aldrich (Oakville, ON, Canada), and KU-55933 from Calbiochem-EMD Biosciences, Inc (La Jolla, CA). siRNA against ATM and LKB1 and negative control siRNA (Alexa Fluor 488) were purchased from QIAGEN (Mississauga, ON, Canada), JC-1 (5,59,6,69-tetramethylbenzimidazolcarbocyanine iodide) from eBiosience (San Diego, CA).

Techniques: Transfection, Incubation, Western Blot