Chk2 inhibitor 2

We cultured mouse adrenocortical tumor Y1 cells in Dulbecco’s modified Eagle medium (DMEM)-F12 medium and human embryonal kidney cells transformed with SV40 large T antigen 293FT cells in DMEM at 37 °C in a humidified atmosphere at 5% CO₂. All media were supplemented with 10% fetal bovine serum. DAPI staining was regularly used to check whether the Y1 and 293FT cells were free of mycoplasma contamination.
For drug treatment, cells were incubated with or without vanillin (DNA-PK inhibitor, V110-4, 1 mM), etoposide (ETO, E1383, at 10, 20, 50, 100, or 200 μM), roscovitine (centrosome inhibitor, R7772, 20 μM), Ku55933 (competitive ATM kinase inhibitor, SML1109, 10 μM), 3-methyladenine (3-MA, autophagy inhibitor, 5142-23-4, 5 mM), UCN-01 (Chk1 selective inhibitor, U6508, 100 nM), and chloroquine (CQ, autophagy inhibitor, 50-63-5, 50 μM), which were purchased from Sigma, St. Louis, MO, USA. Chk2 inhibitor II (Chk2 selective inhibitor, 220,491, 10 μM) was purchased from Merck Millipore, Darmstadt, Germany. Akt inhibitor IV (Akt selective inhibitor, 124,011, 5 µM) was purchased from Cell Signaling, Beverly, MA, USA. Cells were treated with drugs for 24 h, except those treated with ETO were treated for 24, 48, or 72 h.

All cell culture media, supplements and modifying enzymes were purchased from Invitrogen (Karlsruhe, Germany) if not otherwise indicated. Doxorubicin was from Biotrend Chemicals (Cologne, Germany), paclitaxel, and propidium iodide were obtained from Sigma-Aldrich (Taufkirchen, Germany). Gö6983, KU55933, SB203580, U0126 and Chk2 inhibitor II were from Merck (Darmstadt, Germany). The caspase inhibitors Z-VDVAD-FMK and Z-VAD-FMK were derived from R & D Systems (Wiesbaden, Germany). The following antibodies were used in this study: anti-HuR (sc-5261) and anti-Lamin B1 (sc-20682) from Santa Cruz (Heidelberg, Germany), anti-caspase-2 (#611022, BD Biosciences, Heidelberg, Germany), anti-caspase-3 (#9662), anti-caspase-7 (#9494) anti-PARP (#9542) and anti-Bid (#2002) antibodies were from Cell Signaling (Frankfurt, Germany), and anti-β-actin (#A2228) from Sigma-Aldrich. The goat anti-rabbit (sc-2054) and goat anti-mouse (sc-20559) HRP-linked antibodies were from Santa Cruz and Alexa Fluor 488 goat anti-mouse was from Life Technologies (Darmstadt, Germany). Radionucleotides were from Perkin Elmer (Rodgau, Germany) and the ECL system and Hyperfilms were from GE Healthcare (Freiburg, Germany).

RPMI1640 medium, DMEM, TPA, all-trans retinoic acid (ATRA), hemin, etoposide and GW8510 were purchased from Sigma (St. Louis, MO, USA). Chk2 inhibitor II, SB218078 and PD98059 were from Merck (Darmstadt, Germany). λ protein phosphatase was obtained from New England Biolabs (Ipswich, MA, USA). Anti-pRB monoclonal antibody (G3–245), anti-underphosphorylated (under-P) pRB monoclonal antibody (G99–549) and anti-Hsp90 monoclonal antibody (68) were from BD Biosciences (Franklin Lakes, NJ, USA). Anti-Phospho-Chk2 (Thr68) polyclonal antibody and anti-Myc monoclonal antibody (9B11) were from Cell Signaling Technology (Danvers, MA, USA). Anti-Lamin A/C monoclonal antibody (636), anti-Chk2 polyclonal antibody (H-300) and anti-E2F-1 polyclonal antibody (C-20) were from Santa Cruz (Santa Cruz, CA, USA). Phospho-specific pRB monoclonal (pThr356, pSer612 and pSer807) and polyclonal (pThr373, pSer608, pSer780, pSer795, pSer811, pThr821 and pThr826) antibodies were previously described [16] (link)–[21] (link).

U87 cells (ATCC HTB-14), a human glioblastoma cell line, were cultured in minimal essential medium (Gibco) containing 10% fetal bovine serum (FBS), 1% sodium pyruvate, and 2 mM L-glutamine. A549 cells, a human lung carcinoma cell line, were cultured as previously described (Chang et al., 2006 (link)). BE(2)C (ATCC CRL68), a human neuroblastoma cell line were cultured in10% FBS and 2 mM L-glutamine containing RPMI 1640 (Gibco) medium. HEK293T, a human embryonic kidney cells, were cultured in 10% FBS and 2 mM L-glutamine containing Dulbecco's Modified Eagle's Medium (Gibco). Experiments were done using cells with passage 3–5. The ATM inhibitor (KU-55933) was from Calbiochem and the CHK2 inhibitor II was from Merck.

HeLa and HEK293T cells were maintained in DMEM (Hyclone) medium supplemented with 10% fetal bovine serum (Hyclone) and 1% pen-strep (Gibco 15140-122). The CHK2 knockout (KO) HeLa cell line was generated as described previously [17 (link)]. The CHK2 KO cells were transfected with constructs expressing myc-CHK2 (WT or Y156F)-FLAG to establish G418-resistant stable cell lines. These cells were regularly maintained in a medium containing 400 μg/mL G418. The normal human fibroblasts MRC5 and WI38 were maintained in MEM (Gibco) supplemented with 10% fetal bovine serum and 1% pen-strep. All cell lines were from ATCC.
For ionizing radiation treatment, cells were exposed to 8 Gy X-ray using the Torrex 150D inspection system (EG&G) and then incubated for 2 h. Where indicated, cells were treated with CHK2 inhibitor II (C3742, Sigma Aldrich) and JAK2 inhibitor IV (#420139, Calbiochem) at concentrations of 5 and 2.5 μM, respectively. For nocodazole treatment of HeLa and HEK293T (M1404, Sigma Aldrich), either 50 ng/ml (for protein analysis) or 25 ng/ml (for confocal microscopy) was used. For the mitotic arrest of normal fibroblasts WI38 and MRC5, 600 ng/ml of nocodazole was used for protein analysis and 500 n/ml was used for confocal microscopy. Turbofect (Thermo Scientific) and calcium phosphate method were used for transfection of HeLa and HEK293T cells, respectively.

Calcium- and magnesium-free phosphate-buffered saline PBS(−) and paclitaxel (#169-18 611) were purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan). Propidium iodide (PI) and the chk2 inhibitor II were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). SCH900776 was purchased from Med Chem Express (Shanghai, China). The anti-rabbit horseradish peroxidase (HRP)-conjugated IgG (#7074), and the anti-mouse HRP-conjugated IgG (#7076) secondary antibodies, as well as the anti-phospho-cdc2 (Tyr15) (#9111), the anti-cyclin B1 (#4135), the anti-phospho-histone H3 (Ser10) XP (#3377), the anti-phospho-chk1 (Ser296) (#2349), the anti-chk1 (#2360), the anti-phospho-chk2 (Thr68) (#2661), the anti-p21 (#2947) and the anti-β-actin (#4967) monoclonal antibodies were purchased from Cell Signaling Technology Inc (Danvers, MA, USA). The anti-DAP3 (Cat. No. 610662) monoclonal primary antibody was purchased from BD Biosciences (Franklin Lakes, NJ, USA). The Ambion Silencer® Select Pre-designed siRNA against the gene-encoding DAP3 (Cat. No. s1506) and the Silencer® Select Negative #1 Control (Cat. No. AM4611) siRNAs were purchased from Thermo Fisher Scientific, Inc (Waltham, MA, USA).

Vials of cryopreserved PBMCs were thawed at 37°C and washed in ClonaCell-HY Medium A (StemCell Technologies, catalog 03801). EBV was obtained by collecting the supernatant of the marmoset lymphoblastoid cell line (LCL) B95-8 (34 (link)), which was formerly available from the American Type Culture Collection (ATCC) as ATCC CRL-1612. B cells were transformed with EBV by combining washed PBMCs with prepared stocks of filtered B95-8 cell supernatant, using 4.5 mL to transform 8 million to 10 million PBMCs in B cell growth medium, made up of Medium A containing CpG (Invitrogen, oligo ZOEZOEZZZZZOEEZOEZZZT) at the 10 μmol scale (desalted), cyclosporin A (Sigma-Aldrich, catalog C1832), and Chk2 inhibitor II (Sigma-Aldrich, catalog, C3742). Cells were plated at 50 μL/well in a 384-well plate for each suspension of 8 million to 10 million PBMCs. Cells were incubated at 37°C in 7% CO₂ for 6–12 days until LCLs were clearly visible and forming colonies. The plates of transformed B cells were expanded to four 96-well plates in B cell expansion medium (Medium A, CpG, Chk2i II, and 10 million irradiated human PBMCs per plate from an unrelated healthy donor; Nashville Red Cross). The plates were incubated at 37°C in 7% CO₂ for 4–7 days before screening Abs in LCL supernatants for binding to SOSV antigen expressed in cells using a high-throughput flow cytometry assay.