Journal: Oncogenesis

Article Title: DNA-PKcs activates the Chk2–Brca1 pathway during mitosis to ensure chromosomal stability

doi: 10.1038/oncsis.2013.49

Figure Lengend Snippet: DNA-PKcs is required for Chk2 Thr68 phosphorylation during mitosis. ( a ) Human colon cancer HCT116 cells and derivative DNA-PKcs −/− cells were synchronized with nocodazole (50 ng/ml, 16 h) or were irradiated (4 Gy, 1 h). Whole-cell lysates were separated by electrophoresis and western blotted with the indicated antibodies. ( b ) HeLa cells transfected with a control siRNA or with an siRNA against DNA-PKcs were synchronized with nocodazole (50 ng/ml, 16 h) or were irradiated (4 Gy, 1 h). Whole-cell lysates were separated by electrophoresis and were western blotted with the indicated antibodies. Asynchronous (Asy), mitosis enriched (M), γ-ray irradiation (IR).

Article Snippet: Anti-Chk2 total (Cell Signaling, Beverly, MA, USA), anti-phosphorylated Chk2 at Thr68 (Cell Signaling), anti-phospho-histone H3 (EMD Millipore, Billerica, MA, USA), anti-β-actin (Sigma) and anti-Brca1 total (Santa Cruz, Dallas, TX, USA) antibodies were purchased from the indicated vendors.

Techniques: Phospho-proteomics, Irradiation, Electrophoresis, Western Blot, Transfection, Control

Journal: Oncogenesis

Article Title: DNA-PKcs activates the Chk2–Brca1 pathway during mitosis to ensure chromosomal stability

doi: 10.1038/oncsis.2013.49

Figure Lengend Snippet: DNA-PKcs kinase inhibition attenuates mitotic induction of Chk2 phosphorylation. ( a ) Wild-type HCT116 cells were treated with nocodazole for 16 h, followed by a 2 h incubation with DMSO or 10 μ M Nu7441 (Nu). ( b ) HeLa cells were subjected to nocodazole (50 ng/ml, 16 h), followed by a 2 h incubation with DMSO, Nu7441, Ku55933 or both Nu7441 and Ku55933 (left panel). HeLa cells were also irradiated (4 Gy, 1 h) with or without pretreatment with Nu7441 and Ku55933 (right panel).

Article Snippet: Anti-Chk2 total (Cell Signaling, Beverly, MA, USA), anti-phosphorylated Chk2 at Thr68 (Cell Signaling), anti-phospho-histone H3 (EMD Millipore, Billerica, MA, USA), anti-β-actin (Sigma) and anti-Brca1 total (Santa Cruz, Dallas, TX, USA) antibodies were purchased from the indicated vendors.

Techniques: Inhibition, Phospho-proteomics, Incubation, Irradiation

Journal: Oncogenesis

Article Title: DNA-PKcs activates the Chk2–Brca1 pathway during mitosis to ensure chromosomal stability

doi: 10.1038/oncsis.2013.49

Figure Lengend Snippet: Phosphomimetic Chk2 and BRCA1 mutants partially rescue chromosomal instability in DNA-PKcs-deficient cells. ( a ) DNA-PKcs −/− cells were transfected with constructs for the expression of flag-tagged Chk2 (wild-type, T68A, T68D). Expression of exogenous flag-tagged Chk2 and endogenous Chk2 was evaluated by western blot analysis with α-Chk2 antibody. ( b ) Aberrances in mitosis were analyzed in control and cells expressing flag-Chk2 in two independent experiments. ( c ) DNA-PKcs −/− cells were transfected with Brca1 constructs (wild-type, S988A, and S988E). Expression of wild-type and mutant Brca1 was evaluated by western blot. ( d ) Aberrances in mitosis were calculated from two independent experiments. * P <0.05; ** P <0.01.

Article Snippet: Anti-Chk2 total (Cell Signaling, Beverly, MA, USA), anti-phosphorylated Chk2 at Thr68 (Cell Signaling), anti-phospho-histone H3 (EMD Millipore, Billerica, MA, USA), anti-β-actin (Sigma) and anti-Brca1 total (Santa Cruz, Dallas, TX, USA) antibodies were purchased from the indicated vendors.

Techniques: Transfection, Construct, Expressing, Western Blot, Control, Mutagenesis

Journal: Oncogenesis

Article Title: DNA-PKcs activates the Chk2–Brca1 pathway during mitosis to ensure chromosomal stability

doi: 10.1038/oncsis.2013.49

Figure Lengend Snippet: Phosphomimetic Chk2 alleviates dysregulation of microtubule nucleation in DNA-PKcs −/− cells. ( a ) HCT116 and DNA-PKcs −/− cells were treated with nocodazole to disrupt microtubules. Microtubule nucleation and regrowth was monitored at the indicated time points after nocodazole removal. ( b ) The length of the microtubule emanating from the centrosomes was measured (n⩾50). ( c ) HeLa cells expressing wild-type Chk2 or T68A or T68D mutant Chk2 were transfected with control or DNA-PKcs-targeted siRNA. Forty-eight hours after transfection, cells were subjected to microtubule nucleation analysis (n⩾50). * P <0.05; ** P <0.01.

Article Snippet: Anti-Chk2 total (Cell Signaling, Beverly, MA, USA), anti-phosphorylated Chk2 at Thr68 (Cell Signaling), anti-phospho-histone H3 (EMD Millipore, Billerica, MA, USA), anti-β-actin (Sigma) and anti-Brca1 total (Santa Cruz, Dallas, TX, USA) antibodies were purchased from the indicated vendors.

Techniques: Expressing, Mutagenesis, Transfection, Control

Journal: Cancers

Article Title: TP-0903 Is Active in Preclinical Models of Acute Myeloid Leukemia with TP53 Mutation/Deletion.

doi: 10.3390/cancers15010029

Figure Lengend Snippet: Figure 3. TP-0903 induces DNA damage response in TP53 mutant AML cell lines. (A) MV4-11 (R248W), (B) HL-60, and (C) Kasumi-1 cells were treated with DMSO or TP-0903 at indicated concentrations for 4 h. Immunoblotting was performed to determine the expression of pChk1 (Ser345), pChk2 (Thr68), and pH2AX (Ser139/Tyr142). Vinculin served as the loading control for pChk1/Chk1 and pChk2/Chk2 blots. GAPDH served as the loading control for pH2AX/H2AX blots. Immunoblots were quantified against respective loading controls (vinculin or GAPDH) using ImageJ. Blots are representative of 2–3 independent experiments.

Article Snippet: Antibodies against p53 (48818; clone DO-7), p21 (2947; clone 12D1), pH2AX (Ser 139) (80312; clone D7T2V), pH2AX (Ser139/Tyr142) (5438; clone N/A), H2AX (7631; clone D17A3), pAURKA(Thr288)/B(Thr232)/C(Thr198) (2914; clone D13A11), AURKA (91590; clone D3V7T), AURKB (3094; clone N/A), pChk1 (Ser345) (2348; clone 133D3), pChk2 (Thr68) (2197; clone C13C1), Chk1 (2360; clone 2G1D5), Chk2 (6334; clone D9C6), vinculin (13901; clone E1E9V), GAPDH (5174S; clone D16H11), and HRP-conjugate secondary antirabbit (7074) were obtained from Cell Signaling Technology (CST) (Danvers, MA, USA).

Techniques: Mutagenesis, Western Blot, Expressing, Control

Journal: Cancer Research

Article Title: Generation of Replication-Dependent Double-Strand Breaks by the Novel N2-G-Alkylator S23906-1

doi: 10.1158/0008-5472.can-05-3946

Figure Lengend Snippet: Figure 7. Chk2 phosphorylation. Cells were treated with various concentrations of S23906-1 for 6 hours, and phosphorylated Chk2 was analyzed by Western blotting. Membrane was also probed with antibody to h-actin as an internal control.

Article Snippet: After gel transfer, anti-phosphorylated Chk2 antibody (Thr68; Cell Signaling Technology, Beverly, MA) was added for 2 hours and membranes were blotted for 1 hour with a horseradish peroxidase–linked secondary antibody (Cell Signaling Technology).

Techniques: Phospho-proteomics, Western Blot, Membrane, Control