Journal: Frontiers in Plant Science

Article Title: Epigenetic Histone Marks of Extended Meta-Polycentric Centromeres of Lathyrus and Pisum Chromosomes

doi: 10.3389/fpls.2016.00234

Figure Lengend Snippet: Distribution of histone methylation marks in L. sativus analyzed by wide-field fluorescence microscopy . Distributions of methylation marks are shown together with phosphorylation marks at the adjacent sites. (A) H3K4me3 and H3T3ph labeling on metaphase chromosomes. Note that H3K4me3 signals occur preferentially at the chromosome termini and are depleted in primary constrictions and heterochromatin blocks. (B) H3K27me2 (green) and H3S28ph (red) labeling on metaphase chromosomes. The H3K27me2 signals are depleted in primary constrictions and enriched in heterochromatin blocks. (C) Detection of H3K9me2 and H3S10ph on prophase chromosomes. Note that signal is depleted exclusively in heterochromatin blocks whereas centromeric regions show equal or even higher signal intensity than chromosome arms. (D) Detection of H3K9me2 and H3S10ph on metaphase chromosomes. Note the depletion of H3K9me2 signal in primary constriction. (E) Detection of H3K9me2 on metaphase chromosomes upon the treatment with λ-phosphatase. Histone dephosphorylation resulted in stronger H3K9me2 signals in primary constrictions than on chromosome arms. This experiment demonstrated that the depletion of this mark observed on metaphase chromosomes untreated with λ-phosphatase was due to the inability of the antibody to recognize H3K9me2 on histones phosphorylated at adjacent site(s). Bars = 10 μm.

Article Snippet: Histone dephosphorylation was carried out with λ-phosphatase (New England Biolabs) for 1 h at 30°C in 1 × NEB buffer for protein metallophosphatases supplemented with 1 mM MnCl 2 .

Techniques: Methylation, Fluorescence, Microscopy, Phospho-proteomics, Labeling, De-Phosphorylation Assay

Journal: bioRxiv

Article Title: Small molecule inhibitors and a kinase-dead expressing mouse model demonstrate that the kinase activity of Chk1 is essential for mouse embryos and cancer cells

doi: 10.1101/2020.01.21.913467

Figure Lengend Snippet: Chk1 inhibitors reduce expression of Chk1 and induce DNA damage. A) λ820 mouse lymphoma cells were treated with LY2603618 for 24 h and cells were analyzed by immunoblotting for Chk1 and loading control β-actin. B) λ820 mouse lymphoma cells were treated with CCT245737 and cells were analyzed by immunoblotting for apoptosis marker cleaved PARP (cPARP), total or phosphorylated (p)Chk1 and DNA damage marker phosphorylated histone 2AX (γH2Ax). C) λ820 mouse lymphoma cells were treated with LY2603618 or ATR inhibitor VE821 for 24 h and cells were analyzed by immunoblotting for total Chk1, pChk1, phosphorylated (p)RPA2and loading control β-actin.

Article Snippet: Blocked membranes were then blotted with antibodies directed against the following proteins: p-Chk1, cleaved PARP (Cell Signaling Technology, Danvers, MA, USA), Chk1 (Santa Crutz Biotechnology, Dallas, TX, USA), Actin (Sigma-Aldrich) or phosphorylated-histone-H2AX (S139, γH2AX; Merck Millipore).

Techniques: Expressing, Western Blot, Marker

Journal: bioRxiv

Article Title: Small molecule inhibitors and a kinase-dead expressing mouse model demonstrate that the kinase activity of Chk1 is essential for mouse embryos and cancer cells

doi: 10.1101/2020.01.21.913467

Figure Lengend Snippet: Chk1 kinase activity is essential to suppress DNA damage signaling activation and for fibroblast viability. A) Mouse fibroblasts from CreER mice carrying one floxed Chek1 allele and a wildtype (FL/WT), kinase-dead (FL/KD) or another floxed allele (FL/FL) were treated with vehicle or 4-hydroxytamoxifen (4HT) for 4 days and/ or with UVB for 30 s (~700 J/m 2 ), and harvested two hours after radiation or mock. Immunoblotting was performed for Chk1 and γH2Ax. B) Mouse fibroblast expressing CreER, and a floxed and a kinase-dead or wildtype Chek1 allele were transduced with a Myc-expressing retrovirus. Cells were treated with vehicle or tamoxifen and long-term survival was determined by staining cell monolayers. C) Cells manipulated the same as in A were analyzed by immunoplotting for indicated markers of apoptosis (PARP), replication stress (RPA2) and DNA damage (γH2Ax).

Article Snippet: Blocked membranes were then blotted with antibodies directed against the following proteins: p-Chk1, cleaved PARP (Cell Signaling Technology, Danvers, MA, USA), Chk1 (Santa Crutz Biotechnology, Dallas, TX, USA), Actin (Sigma-Aldrich) or phosphorylated-histone-H2AX (S139, γH2AX; Merck Millipore).

Techniques: Activity Assay, Activation Assay, Western Blot, Expressing, Transduction, Staining

Journal: Cancer Research

Article Title: Stalled Replication Induces p53 Accumulation through Distinct Mechanisms from DNA Damage Checkpoint Pathways

doi: 10.1158/0008-5472.can-05-1790

Figure Lengend Snippet: Figure 3. Induction of p53 by hydroxyurea correlates with the activation of MRN-ATM pathway but is insensitive to caffeine. A, ATM is autophosphorylated after DNA damage and replication block. Normal lymphoblastoid cells (WT) were treated with the indicated agents for 24 hours. The levels of phospho-ATMSer1981 and total ATM were detected by immunoblotting. Extracts of ataxia telangiectasia lymphoblastoid cells act as negative controls. B, hydroxyurea induces significantly fewer DNA breakage than Adriamycin. Wild-type lymphoblastoid cells were mock treated or treated with hydroxyurea or Adriamycin for 16 hours. The cells were then harvested for single-cell gel electrophoresis assay. Representational images are shown and tail movements were quantified. Columns, mean of three independent experiments; bars, SD. C, hydroxyurea-induced ATM, p53, and histone H2AX phosphorylation are not suppressed by caffeine. Wild-type lymphoblastoid cells were treated with buffer or hydroxyurea for 6 hours before buffer or caffeine was added for another 6 hours. Cell extracts were prepared and the expression of p53, phospho-p53Ser20, phospho-ATMSer1981, ATM, phospho-histone H3Ser10, and phospho-histone H2AXSer139 was detected by immunoblotting. Uniform loading of lysates was confirmed by immunoblotting for CDC2.

Article Snippet: Polyclonal antibodies against Ser139-phosphorylated histone H2AX were either gifts from Junjie Chen (Mayo Clinic, Rochester, MN) or obtained from Bethyl Laboratories (Montgomery, TX).

Techniques: Activation Assay, Blocking Assay, Western Blot, Single Cell Gel Electrophoresis, Phospho-proteomics, Expressing