Journal: PloS one

Article Title: A high through-put screen for small molecules modulating MCM2 phosphorylation identifies Ryuvidine as an inducer of the DNA damage response.

doi: 10.1371/journal.pone.0098891

Figure Lengend Snippet: Figure 1. Characterization of pSer40/41MCM2 antibody. A) HeLa whole cell extract was incubated with Lambda phosphatase in the presence or absence of phosphatase inhibitors. Proteins were analyzed by western blot with the indicated antibodies. B) Equal amounts of protein extract were separated on a single SDS-PAGE gel and transferred onto membranes. Vertical slices of membrane (each with identical protein content) were then incubated with anti-pSer40/41MCM2 antibody in the presence of the indicated competitor peptides (upper panels). Membranes were then re-probed with an anti-MCM2 antibody (lower panels). Identical exposures are shown. C) HeLa cells growing on coverslips were fixed and stained with anti- pSer40/41MCM2 antibody in the presence of the indicated competitor peptides. DNA was counterstained with DAPI. D) Serial sections of normal and cancerous colon tissue were used in IHC with the pSer40/41MCM2 antibody in the presence or absence of competitor peptide. Nuclear positivity is shown by brown color. E) HeLa cells were treated with PHA-767491 for the indicated time. Protein extracts were prepared and analysed by western blot with the indicated antibodies. doi:10.1371/journal.pone.0098891.g001

Article Snippet: Cell culture HeLa S3 (CCL-2.2) cells used for the screening and cell based assays were authenticated by LGC standards at the time of the screening [5], U2OS [30] and human foreskin fibroblasts (HFF) [31] cells were cultured in Dulbelcco’s modified Eagle’s medium (DMEM) supplemented with heat inactivated 10% fetal bovine serum and 1% penicillin-streptomycin (Sigma-Aldrich).

Techniques: Incubation, Western Blot, SDS Page, Membrane, Staining

Journal: PloS one

Article Title: A high through-put screen for small molecules modulating MCM2 phosphorylation identifies Ryuvidine as an inducer of the DNA damage response.

doi: 10.1371/journal.pone.0098891

Figure Lengend Snippet: Figure 3. Confirmation and in vitro characterization of Hit compounds. A) HeLa cells were treated with the ten compounds that produced the strongest reduction in pSer40/41MCM2 levels in the primary screen. Protein extracts were prepared and levels of residual pSer40/41MCM2 phosphorylation were measured by western blot analysis with the indicated antibodies. B) The same compounds were tested for their ability to inhibit CDC7 kinase activity in in vitro kinase assay. Kinase reactions were performed on a synthetic MCM2 substrate in the absence or presence of the indicated drug and reactions were run on SDS-PAGE gels. CDC7 activity on the synthetic MCM2 substrate was monitored by Western blotting with an anti-pSer108MCM2 antibody. As a control the levels of CDC7 kinase and synthetic MCM2 substrate present in each reaction were also assessed and shown to be similar in all reactions. doi:10.1371/journal.pone.0098891.g003

Article Snippet: Cell culture HeLa S3 (CCL-2.2) cells used for the screening and cell based assays were authenticated by LGC standards at the time of the screening [5], U2OS [30] and human foreskin fibroblasts (HFF) [31] cells were cultured in Dulbelcco’s modified Eagle’s medium (DMEM) supplemented with heat inactivated 10% fetal bovine serum and 1% penicillin-streptomycin (Sigma-Aldrich).

Techniques: In Vitro, Produced, Phospho-proteomics, Western Blot, Activity Assay, Kinase Assay, SDS Page, Control

Journal: PloS one

Article Title: A high through-put screen for small molecules modulating MCM2 phosphorylation identifies Ryuvidine as an inducer of the DNA damage response.

doi: 10.1371/journal.pone.0098891

Figure Lengend Snippet: Figure 4. Ryuvidine and Mitoxantrone reduce CDC7 levels and pSer40/41 MCM2 phosphorylation. A) Molecular structure of PHA- 767491, Ryuvidine and Mitoxantrone. B) HeLa cells were incubated with either CDC7 inhibitor PHA-767491, Ryuvidine or Mitoxantrone at 10 micromolar each for the indicated time. Protein extracts were prepared and analyzed by western blot with the indicated antibodies. C) Levels of Cdc7-independent pSer41MCM2 and Cdc7-dependent pSer40/41MCM2 phosphorylation assessed in HeLa cells treated with the indicated concentration of PHA-767491, Ryuvidine or Mitoxantrone. D) Cells were incubated with either PHA-767491, Ryuvidine or Mitoxantrone at the indicated concentration for nine hours. Protein extracts were then prepared and analyzed by western blot with the indicated antibodies. E) HeLa cells incubated with either PHA-767491, Ryuvidine or Mitoxantrone at 10 micromolar each for nine hours in the presence or absence of 20 micromolar of caspase inhibitor Boc-D-fmk. Levels of Ser40/41MCM2 phosphorylation, CDC7 and MCM2 abundance as well as PARP cleavage, were assessed by western blotting. doi:10.1371/journal.pone.0098891.g004

Article Snippet: Cell culture HeLa S3 (CCL-2.2) cells used for the screening and cell based assays were authenticated by LGC standards at the time of the screening [5], U2OS [30] and human foreskin fibroblasts (HFF) [31] cells were cultured in Dulbelcco’s modified Eagle’s medium (DMEM) supplemented with heat inactivated 10% fetal bovine serum and 1% penicillin-streptomycin (Sigma-Aldrich).

Techniques: Phospho-proteomics, Incubation, Western Blot, Concentration Assay

Journal: PloS one

Article Title: A high through-put screen for small molecules modulating MCM2 phosphorylation identifies Ryuvidine as an inducer of the DNA damage response.

doi: 10.1371/journal.pone.0098891

Figure Lengend Snippet: Figure 5. Ryuvidine and Mitoxantrone block DNA synthesis. A) HeLa cells growing on coverslips were incubated for one hour with either Ryuvidine, Mitoxantrone or PHA-767491. 15 minutes before the end of treatment EdU was added and cells were fixed. DNA synthesis was revealed by covalently linking 6-carboxyfluorescein TEG-Azide to incorporated EdU and fluorescence microscopy. Nuclei were stained with DAPI. Representative fields are shown. B) HeLa cells were either mock treated (DMSO) or treated for the indicated times with PHA- 767491 or Ryuvidine. Fifteen minutes before the end of treatment EdU was added to the medium. DNA synthesis was analyzed by EdU incorporation assay and Flow Cytometry. Percentage of EdU positive cells at the indicated times after addition of the drugs is reported. doi:10.1371/journal.pone.0098891.g005

Article Snippet: Cell culture HeLa S3 (CCL-2.2) cells used for the screening and cell based assays were authenticated by LGC standards at the time of the screening [5], U2OS [30] and human foreskin fibroblasts (HFF) [31] cells were cultured in Dulbelcco’s modified Eagle’s medium (DMEM) supplemented with heat inactivated 10% fetal bovine serum and 1% penicillin-streptomycin (Sigma-Aldrich).

Techniques: Blocking Assay, DNA Synthesis, Incubation, Fluorescence, Microscopy, Staining, Flow Cytometry

Journal: PloS one

Article Title: A high through-put screen for small molecules modulating MCM2 phosphorylation identifies Ryuvidine as an inducer of the DNA damage response.

doi: 10.1371/journal.pone.0098891

Figure Lengend Snippet: Figure 6. Ryuvidine elicits a DNA damage response. A) Protein extracts prepared from HeLa cells treated with either PHA-767491, Ryuvidine, Mitoxantrone or hydroxyurea (HU) for the indicated time were analyzed by western blot with indicated antibodies. B) Formation of c-H2AXnuclear foci in cells treated with ATM inhibitor KU55933, Mitoxantrone, Ryuvidine alone or in combination was assessed by fluorescence microscopy. Nuclei were stained with DAPI. Representative fields are shown. doi:10.1371/journal.pone.0098891.g006

Article Snippet: Cell culture HeLa S3 (CCL-2.2) cells used for the screening and cell based assays were authenticated by LGC standards at the time of the screening [5], U2OS [30] and human foreskin fibroblasts (HFF) [31] cells were cultured in Dulbelcco’s modified Eagle’s medium (DMEM) supplemented with heat inactivated 10% fetal bovine serum and 1% penicillin-streptomycin (Sigma-Aldrich).

Techniques: Western Blot, Fluorescence, Microscopy, Staining