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: Journal of Virology

Article Title: The Human Papillomavirus E6 PDZ Binding Motif Links DNA Damage Response Signaling to E6 Inhibition of p53 Transcriptional Activity

doi: 10.1128/JVI.00465-18

Figure Lengend Snippet: Different DDR pathways lead to phosphorylation of E6 in response to different stresses. (A to C) HeLa cells were treated with specific inhibitors of ATR (ATRi), ATM (ATMi), Chk1 (Chk1i), or Chk2 (Chk2i) for 15 h and then with dimethyl sulfoxide (DMSO) as a control (A) or treated with nocodazole (B) or H2O2 (C). Western blots were probed with antibodies against total HPV-18 E6 and HPV-18 E6 phospho-T156. α-Actinin was used as a loading control. (D) The assay was repeated with H2O2, additionally treating the cells with a specific PKA inhibitor, H89, as well as Chk1i and Chk2i. The pE6 levels were detected by Western blotting, and total E6 is also shown. α-Actinin was used as a loading control. (E) HeLa cells were treated with H2O2, together with the specific PKA inhibitor H89 and the DDR kinase inhibitors, as indicated. The pChk2 levels were analyzed to check the efficacy of ATMi and ATRi, and pE6 levels were also analyzed in the presence or absence of the various inhibitors. Also shown are total E6 levels. α-Actinin was used as a loading control.

Article Snippet: The antibodies used were as follows: rabbit anti-α-actinin (Santa Cruz), mouse monoclonal anti-p53 (Santa Cruz), and rabbit polyclonal pChk2 (Thr68) (Novus Biologicals).

Techniques: Phospho-proteomics, Control, Western Blot