Journal: Oncotarget

Article Title: YM155 potently triggers cell death in breast cancer cells through an autophagy-NF-kB network

doi:

Figure Lengend Snippet: A. S139-phosphorylated-H2AX and Survivin proteins expression were performed by immunoblot analysis in YM155 treated-cells using the p53-mutated MDA-MB231 cell line and the non mutated-p53 cell line Cal51. P53, its S15 phosphorylated form, and its gene target p21 were also assessed in these latter p53 proficient cells. B. Initiation of DNA repair was evaluated by counting 53BP-1 nuclear foci formed in GFP-53BP-1-expressing Cal51 after indicated treatment. Cisplatin (50 μM) was used as positive control. Representative images of flourescent cells in each conditions are also inserted in the figure. C. Phosphorylation of Chk1 and Chk2 on S317 and T68 respectively, upon YM155 treatment was evaluated by immunoblot analysis in comparison with untreated Cal51 cells. D. Cell cycle analysis was performed in cell lines using IP staining and flow cytometry analysis after 48 h incubation with YM155 compared to untreated control cells.

Article Snippet: Antibodies against Survivin was purchased from R&DSystems (Lille, France), antibodies against LC3, p21, pS15-p53, pS317-Chk1, pT68-Chk2, IKK2, Actin, γH2AX from Merck Millipore (Saint-Quentin en Yvelines, France), antibodies against HSP90, p53 and cleaved (i.e. activated) caspase3 from Becton Dickinson (Pont de Claix, France), antibody against BAX from Dako (Courtaboeuf, France), and antibody against p62 from Santa Cruz (Heidelberg, Germany).

Techniques: Expressing, Western Blot, Positive Control, Cell Cycle Assay, Staining, Flow Cytometry, Incubation

Journal: Experimental Neurobiology

Article Title: KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells

doi: 10.5607/en25017

Figure Lengend Snippet: KCC-07 suppresses growth of U-87MG and SH-SY5Y cell lines. (A) Dose dependent proliferation responses of U-87MG and SH-SY5Y cell lines to KCC-07 as measured by MTT assay. Both U-87MG (glioma cell line) and SH-SY5Y (neuroblastoma cell line) are sensitive to KCC-07 treatment in a dose dependent manner. Representative data are shown in the graphs (n=12). The readouts of the NT group were set as 100%, then the survival rates were calculated as a relative values to the NT group. Each experiment was repeated twice independently. (B) Dose dependent proliferation responses of U-87MG and SH-SY5Y cell lines to DNA damage induced by either phleomycin (Phleo) or etoposide (ETP) in combination with KCC-07 exposure as measured by MTT assay. Dose dependency of DNA damage inducing drugs is not affected by KCC-07 treatment. Representative data are shown in the graphs (n=4~6). The readouts of the NT group were set as 100%, then the survival rates were calculated as a relative values to the NT group. Each experiment was repeated twice independently. (C) Detection of p53 stabilization in the nucleus following KCC-07 treatment and DNA damage induction, indicating the activation of p53 signaling in both U-87MG and SH-SY5Y cell lines. The distribution of MDM2, an E3 ubiquitin ligase for p53, was not changed; its localization is restricted to the cytosol. Representative data are shown in the figure. Each experiment was repeated three times independently. The experimental conditions of drug treatments are indicated in the figure. NT, Not treated; NS, not significant; C, cytosolic fraction; N, nuclear fraction.

Article Snippet: The antibodies used for Western blot analysis were ATM (Abcam, Cambridge, UK), ATM-pS1981 (Cell Signaling Technology, MA, USA), KAP1 (Novus Biologicals, CO, USA), KAP1-pS824 (Novus Biologicals), CHK2 (Millipore, MA, USA), CHK2-pT68 (Novus Biologicals), γ-H2AX (Cell Signaling Technology), p53 (Santa Cruz Biotechnology, TX, USA), p53-pS15 (Cell Signaling Technology), MDM2 (Santa Cruz Biotechnology), GAPDH (Genetex, CA, USA), and histone H3 (Abcam).

Techniques: MTT Assay, Activation Assay, Ubiquitin Proteomics

Journal: Experimental Neurobiology

Article Title: KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells

doi: 10.5607/en25017

Figure Lengend Snippet: Both KCC-07 and DNA damage induction result in known p53 dependent gene expression. (A) Induction of CDKN1A expression, which is p53 dependent and involved in cell cycle regulation, by either KCC-07 or DNA damaging reagent treatments measured by real-time PCR. Combined treatment with KCC-07 and DNA damaging reagents did not further increase CDKN1A expression in both cell lines. The SH-SY5Y cell line is more sensitive to DNA damaging reagents. The gene expression levels were normalized by β-actin real-time PCR readout. Representative data are shown in the graphs (n=3). Each experiment was repeated twice independently. (B) Induction of BBC3 expression, which is p53 dependent and involved in apoptosis, by DNA damaging reagents. KCC-07 treatment did not induce BBC3 expression in both cell lines. The gene expression levels were normalized by β-actin real-time PCR readout. Representative data are shown in the graphs (n=3). Each experiment was repeated twice independently. (C) Consistent DNA damage response (DDR) following combined treatment with KCC-07 and DNA damaging reagents analyzed by Western blots. Both cell lines displayed well-established DDR including ATM phosphorylation, KAP1 and CHK2 phosphorylation (ATM-dependent), and p53 activation (ATM-dependent) followed by induction of apoptotic and cell cycle arrest factors (p53-dependent, see B and C in this figure) upon DNA damage induction by Phleo or ETP treatments. KCC-07 addition does not change the proper DDR upon DNA damage, but sustained H2AX phosphorylation indicates defective DNA damage repair. Ponceau S staining and GAPDH western blot served as loading controls. Representative data are shown. Each experiment was repeated twice independently. The experimental conditions of drug treatments are indicated in the figure. NS, not significant.

Article Snippet: The antibodies used for Western blot analysis were ATM (Abcam, Cambridge, UK), ATM-pS1981 (Cell Signaling Technology, MA, USA), KAP1 (Novus Biologicals, CO, USA), KAP1-pS824 (Novus Biologicals), CHK2 (Millipore, MA, USA), CHK2-pT68 (Novus Biologicals), γ-H2AX (Cell Signaling Technology), p53 (Santa Cruz Biotechnology, TX, USA), p53-pS15 (Cell Signaling Technology), MDM2 (Santa Cruz Biotechnology), GAPDH (Genetex, CA, USA), and histone H3 (Abcam).

Techniques: Gene Expression, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Phospho-proteomics, Activation Assay, Staining