Ku55933

The ATM inhibitors KU-55933 (S1092) and CP-466722 (S2245) were obtained from Selleckchem. LOPAC-1280 library, Nutlin-3 and Etoposide were purchased from Sigma-Aldrich.
Screening of the LOPAC small compound library was performed as follows: 10 000 cells per well were seeded in 96-well flat bottom plates (Corning CellBind CLS3340-50EA), 24 hours later cells were treated with drugs for 24 hours at a final concentration of 5μM in 0.5% DMSO in the presence of 0.5μM Etoposide, or with 0.5μM Etoposide in drug-free DMSO (negative control) or with DMSO only. Cells were then fixed for 10 minutes in 10% neutral buffered formalin solution (Sigma HT5012), nuclei stained for 10 minutes with DAPI (1μg/ml in PBS) and stored at 4°C until scanned (Thermo Cellomics Compartmental Analysis V4). Acquisitions were made with a 20x objective, both with XF93-Hoechst filter, 0.05sec exposure (DAPI) and XF93-TRITC filter, 0.5sec (RFP).

Two percent sodium alginate was prepared by dissolving sodium alginate (Sigma-Aldrich) in phosphate-buffered saline (PBS) and sterilizing by heating to 80°C in a water bath for 15 min (Leo et al., 1990 (link)). Anti-CD150-PE, anti-GRPC5C Alexa Fluor-405, and anti-Ki67-Alexa Fluor 405 antibodies for flow cytometry were purchased from R&D Systems. Anti-CD49f-PE and anti-CD90-VioBlue were purchased from Miltenyi Biotec. Transforming growth factor beta-1 (TGFβ-1) and fibroblast growth factor were obtained from PeproTech. Fms-like tyrosine kinase-3 (Flt-3), G-CSF, stem cell factor (SCF), and interferon alpha were purchased from ImmunoTools. All-trans retinoic acid (ATRA) was obtained from Sigma-Aldrich. Hoechst 33342, propidium iodide (PI), and pyronin Y (PY) were purchased from Sigma-Aldrich. The CountBright^TM Absolute Counting Beads was purchased from Thermo Fisher Scientific. MHY1485, AZD8055, rosiglitazone, KU-55933, LEE011, roscovitine (seliciclib, CYC202), glasdegib (PF-04449913), sodium butyrate, dasatinib, BIO, plerixafor (AMD3100), quizartinib, and sorafenib were purchased from Selleckchem. Adiponectin, triglitazone LE135, PD169316, harmine, nilotinib, ethylisopropyl amiloride, anisomycin, and curcumin were purchased from Sigma-Aldrich, while prostaglandin E2 was from BioVision/Cambridge Bioscience. Cytarabine and daunorubicin were purchased from Sigma-Aldrich.

Immortalized human foreskin keratinocytes (HFK), provided by Michael Underbrink (University of Texas Medical Branch, Galveston, TX, USA), were grown in EpiLife medium (Gibco, Gaithersburg, MD, USA), supplemented with 60 µM calcium chloride (Gibco), human keratinocyte growth supplement (Gibco), and 1% penicillin-streptomycin (Caisson, Smithfield, UT, USA). U2OS and HCT116 cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. Zeocin (Alfa Aesar, Ward Hill, MA, USA) and H2O2 were used to induce DSBs. NU7441 (Selleckchem) was used to inhibit DNA-PKcs phosphorylation. KU55933 (Selleckchem, Houston, TX, USA) was used to inhibit ATM kinase activity.

129/Sv × C57BL/6 ES cells were cultured on feeder cells using standard ES cell culture conditions. Cells were transfected with px330 expressing Cas9, mCherry, and sgRNA using Lipofectamine 3000 Reagent (Invitrogen) according to the manufacturer’s instructions. Forty-eight hours after transfection, mCherry-positive ES cells were sorted into 96 wells using BD FACS AriaII for further culturing. After 7 days of culturing, the colonies were picked up and expanded for further analysis.
For cell treatment with drugs, the ATM inhibitor KU-55933 (number S1092, Selleckchem) was used at 20 μM. Cells were transfected with plasmids (pX330-mCherry-Ssty2-A and B) and mCherry-positive mouse ES cells were sorted by FACS 12 h after transfection. DNA-FISH analysis was performed 24 or 48 h later.
Human iPSCs were purchased from ATCC (ATCC® ACS-1003™) and cultured on irradiated mouse embryonic fibroblast (iMEFs) feeder layers in serum-free N2B27-LCDM medium as described previously [44 (link)]. For transfection, cells were dissociated using TrypLE, replated in iMEF-coated 12-well plates, and transfected in suspension with gRNAs, Cas9, and EGFP or mCherry plasmid using Lipofectamine 3000. Twenty-four hours after transfection, EGFP⁺/mCherry⁺ cells were sorted and used for DNA-FISH analysis at 7 days post-transfection.

Baf A1, CQ and LA were purchased from Sigma (Deisenhofen, Germany). The AKT1/2-specific inhibitor AKTVIII was obtained from Merck Millipore (Darmstadt, Germany). The ATM inhibitor KU-55933, the AKT2-specific inhibitor CCT128930 and the pan-caspase inhibitor Z-VAD-FMK were from Selleck Chemicals (Houston, TX, USA).

Immunofluorescence (IF) staining was performed following our published conditions [64 (link),65 (link),66 (link)]. Briefly, cells were fixed with prechilled (−20 °C) acetone–methanol for 15 min prior to the addition of primary antibodies anti-γH2AX (1:100, Cell Signaling, Danvers, MA, USA) and anti-phospho-ATM (S1981) (1:100, Cell Signaling) at 4 °C overnight. After rinsing, FITC-Donkey anti-mouse IgG (1:200, Jackson Immuno Research Lab, West Grove, PA, USA) and Rhodamine-Donkey anti-rabbit IgG (1:200, Jackson Immuno Research Lab) were added for 1 h at room temperature. Slides were mounted using the VECTASHIELD mounting medium containing DAPI (VECTOR Lab Inc., Burlington, ON, Canada). VE-281 (Selleckchem, Burlington, ON, Canada) or KU-55933 (Selleckchem) was added to cells 8 h prior to treatment. Images were then acquired with a fluorescent microscope (Axiovert 200, Carl Zeiss, North York, ON, Canada).