Roscovitine

Roscovitine (sc‐24002) and RO‐3306 (sc‐358700) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Recombinant t‐Darpp was used as a substrate for CDK1 and CDK5 phosphorylation. CDK1 (C22‐18G) and CDK5 (C33‐10G‐05) were purchased from Signal Chem Pharmaceuticals (Richmond, BC, Canada). CDK1 or CDK5 was diluted to a final concentration of 5 μg·mL⁻¹ in kinase dilution buffer (K23‐09; Signal Chem Pharmaceuticals). The reactions were preincubated with t‐Darpp for 10 min at 30 °C. The kinase reaction was initiated by the addition of 200 μm ATP (final concentration) and continued at 30 °C over a period of 60 min. Reactions were quenched by the addition of sample buffer and boiled for 5 min. RO‐3306 or Roscovitine at 10 μm was used to inhibit CDK1 or CDK5 activity, respectively. These inhibitors were added directly to the kinase reaction during the preincubation period. Samples were analyzed by western blot analysis as described below.

Seven days after plating, neurons were incubated in neurobasal medium containing either 10mM or 20mM of KCl for 6 hours (h) to induce hyperexcitability. Control neurons were incubated in neuronal medium containing 5mM of KCl for 6 h. Then, the medium was collected and the cells lysed. To block the fragmentation of the Golgi induced by elevated concentrations of KCl, neurons were pre-incubated either with 10μM of olomoucine (Cayman) or 2μM of roscovitine (Santa Cruz) for 1 h.

Generation of laser-induced DSB and recruitment of fluorescence protein-tagged proteins were performed with a SRS NL100 nitrogen MicroPoint system equipped to a Nikon Eclipse TE2000 spinning disk confocal microscope with Volocity software 6.3 (Perkin-Elmer)^{34 (link),64 (link)}. For treatment of CDK inhibitors, U2OS cells expressing GFP/YFP-tagged RECQL4 and YFP-MRE11 were pre-incubated with DMSO, 20 µM Roscovitine (Santa Cruz), 10 µM CDK1 inhibitor RO3306, 10 µM CDK2 inhibitor 2-III (EMD Millipore) for 4 h under standard cell culture conditions, and then subjected to micro point laser-irradiation. To study the function of CDK-dependent phosphorylation of Ser89 and Ser251 on RECQL4 recruitment to DSBs, GFP-tagged WT RECQL4, RQ4-2A, and RQ4-2D were expressed by transfecting the plasmids pEGFP-C1-RQ4Wt, pEGFP-C1-RQ4-2A, and pEGFP-C1-RQ4-2D into U2OS cells, respectively. For CDK 1and CDK2 inhibition, the U2OS cells were incubated with RO3306 and CDK2 inhibitor-III for 4 h before laser irradiation. The recruitment of these proteins was conducted as described above. The fluorescence intensity of the damaged area was measured with Volocity imaging software and normalized to that of a control area. The results are presented as mean ± s.e.m., and P -values were measured with Student’s t-test.

Ox-1 (Santa Cruz), ZM (ACC), blebbistatin (APEXBIO), ABT-263 (Selleckchem), Roscovitine (Santa Cruz), RO-3306 (Santa Cruz), were dissolved in dimethyl sulfoxide (DMSO) and stored at −20°C. Human NB4 and U937 cell lines were obtained from the American Type Culture Collection (ATCC) and grown in RPMI 1640 medium supplemented with 10% FBS. Cells were cultured at 37°C in a humidified atmosphere containing 5% CO₂. Control cultures received an equivalent amount of DMSO only. Bone marrow mononuclear cells (BMMCs) or mobilized peripheral blood mononuclear cells (PBMCs) were obtained from 10 newly diagnosed AML patients and 3 healthy donors. All donors provided written informed consent, and the study had the approval of the Institute Research Ethics Committee at Sun Yat-Sen University, in accordance with the Declaration of Helsinki. Patient characteristics are shown in Table 1. PBMCs and BMMCs were enriched from the diagnostic bone marrow samples of patients with de novo AML by Ficoll-Hypaque (Sigma-Aldrich, St Louis, MO) density gradient centrifugation.