Journal: Biological & pharmaceutical bulletin

Article Title: Glionitrin A, a new diketopiperazine disulfide, activates ATM-ATR-Chk1/2 via 53BP1 phosphorylation in DU145 cells and shows antitumor effect in xenograft model.

doi: 10.1248/bpb.b13-00719

Figure Lengend Snippet: Fig. 2. Glionitrin A Elevates H2A.X Phosphorylation by Responding to DNA Damage Resulting from the Activation of ATM/ATR-Chk1/2 in a 53BP1-Dependent Manner

Article Snippet: Primary antibodies against phospho-H2A.X (Ser139), phospho-53BP1 (S11778), 53BP1, phospho-ATM (Ser1981), phospho-ATR (Ser428), ATR, phospho-Chk1 (Ser317), phospho-Chk1 (Ser345), Chk1, phospho-Chk2 (Thr68), phosphoChk2 (Thr387), Chk2, cdc25A, phosphor-cdc2 (Tyr15), cdc2, cleaved caspase-8, Bid, Bax, cleaved caspase-9, cleaved caspase-3, poly(ADP-ribose) polymerase (PARP), apoptosisinducing factor (AIF), endonuclease G (EndoG), histone deacetylase 1 (HDAC1), cytochrome c oxidase subunit IV (COX IV) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and peroxidase-conjugated secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, U.S.A.).

Techniques: Phospho-proteomics, Activation Assay

Journal: Cell reports

Article Title: Nascent DNA Proteomics Reveals a Chromatin Remodeler Required for Topoisomerase I Loading at Replication Forks.

doi: 10.1016/j.celrep.2016.03.027

Figure Lengend Snippet: Figure 2. Transcription Is a Major Determinant of Replication Hindrance by CPT (A) Western blot analysis of Chk1 phosphorylation on Ser345 in HeLa S3 cells exposed to CPT (1 mM) for the indicated time. When indicated, cells were pre- treated for 3 hr with 50 mM cordycepin, 4 hr with 50 mg/ml a-amanitin, or 2 hr with 100 mM DRB. (B) HeLa S3 and HDF cells were pre-treated with transcription inhibitors as described above, and they were labeled with IdU (30 min) and then with CldU (30 min) in the presence of 1 mM CPT when indicated. Graphic representation shows the ratios of CldU versus IdU track length. The horizontal bar represents the median, indicated in red, from at least 50 replication tracks per experimental condition. Differences between experiments were assayed using Mann-Whitney rank test; p values are indicated. (C) Frequency of reversed forks detected by electron microscopy (EM) in U2OS cells. Electron micrographs of a normal replication fork in non-treated cells and a regressed fork from CPT-treated cells are shown. Cells were treated for 1 hr with 25 nM CPT and pretreated or not for 3 hr with 50 mM cordycepin. The total number of analyzed molecules is given in brackets. Above each column, the percentage of reversed forks is indicated. Parental duplex, P; daughter duplex, D; regressed arm, R; NT, non-treated.

Article Snippet: Antibodies against the following proteins were used: BAZ1B (Bethyl Laboratories, A300-446A), SMARCA5 (Bethyl Laboratories, A301-017A), Ser345 Phospho-Chk1 (Cell Signaling Technology, 2348), Chk1 (Santa Cruz Biotechnology, sc-8408), PCNA (Sigma-Aldrich, P8825), H3 (Santa Cruz Biotechnology, sc-10809) Tubulin (GeneTex, GTX27749), Ser4/8 Phospho RPA32 (Bethyl Laboratories, A300-245A), RPA32 (Calbiochem, NA18), and Top1 (BD Pharmingen, 556597).

Techniques: Western Blot, Phospho-proteomics, Labeling, MANN-WHITNEY, Electron Microscopy

Journal: Lung cancer (Amsterdam, Netherlands)

Article Title: CHK1 levels correlate with sensitization to pemetrexed by CHK1 inhibitors in non-small cell lung cancer cells

doi: 10.1016/j.lungcan.2013.09.010

Figure Lengend Snippet: Relationship of CHK1 mRNA expression with patient survival, tumor differentiation and genomic complexity. (A) Analysis of gene expression of 442 lung adenocarcinomas using Kaplan–Meier survival curve shows that patients with high CHK1 mRNA expressing tumors demonstrate an overall poorer 5 year survival, comparing the higher 1/3 patients vs lower 2/3 patients based on CHK1 mRNA levels (log-rank test, p < 0.001). (B) This was verified in an independent cohort of patients with 101 lung adenocarcinomas with CHK1 mRNA verified by RT-PCR (p = 0.02). (C) Tumors with higher CHK1 mRNA expression are more likely to display a poorly-differentiated phenotype as compared to low CHK1 expressing tumors (p < 0.0001) in 442 lung adenocarcinomas. (D) This was verified in the same independent cohort of patients with 101 lung adenocarcinomas (p = 0.0003). CHK1 mRNA expression in lung adenocarcinomas also correlates with an increased number of genomic alterations, specifically gene mutations or amplifications. Tumors were classified into high, medium and low groups based on CHK1 mRNA expression (CHK1 mRNA >100, 100–50, and <50 as cutoff for these 3 groups). (E) Higher CHK1 mRNA expression correlated with an increased number of gene mutations in 54 lung adenocarcinomas (Pearson correlation, r = 0.993, p < 0.001, the mean mutation ±SD are 7.28 ±10.4, 4.23 ±3.7 and 1.14 ±0.99 for high, medium and low groups, respectively). (F) In a separate data-set of 93 lung adenocarcinomas, higher CHK1 mRNA correlated with DNA amplification/gain (Pearson correlation, r = 0.99, p < 0.001, mean amp/gain ±SD are 3.73 ±3.2, 2.29 ±2.1, and 1.0 ±0.76 for high, medium and low groups, respectively). (G) CHK1 mRNA levels do not correlate with gene deletions or LOH for the same 93 tumors.

Article Snippet: PathScan CHK1 Sandwich ELISA kit (Cell Signaling) was used according to manufacturer’s protocol.

Techniques: Expressing, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Mutagenesis, DNA Amplification

Journal: Lung cancer (Amsterdam, Netherlands)

Article Title: CHK1 levels correlate with sensitization to pemetrexed by CHK1 inhibitors in non-small cell lung cancer cells

doi: 10.1016/j.lungcan.2013.09.010

Figure Lengend Snippet: (A) Gene expression analysis for CHK1 mRNA levels in representative sample of 15 cultured lung cancer cell lines and in nontransformed lung cells (NLC). Based on these results, we selected two apparent under-expressing cell lines, H1993 and H1437, and two over-expressing cell lines, H23 and H1299, for further analysis. (B) Relative expression of CHK1 to β-actin by qRT-PCR in selected NSCLC cell lines. Data shown is the aggregate of three independent assays, and suggests that H1437 is actually a CHK1 over-expressing cell line. CHK1 protein levels in selected NSCLC cell lines by Western Blot (C) and by ELISA (D) confirming the three higher CHK1 expressing cell lines (H23, H1437, and H1299) and the one lower expressing line (H1993). (E) CHK1 expression by immunohistochemistry in two representative cell lines (H1993 and H1299) showing increased nuclear CHK1 protein expression (brown stain) in the H1299 cells compared to the H1993 cells. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: PathScan CHK1 Sandwich ELISA kit (Cell Signaling) was used according to manufacturer’s protocol.

Techniques: Gene Expression, Cell Culture, Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Staining

Journal: Lung cancer (Amsterdam, Netherlands)

Article Title: CHK1 levels correlate with sensitization to pemetrexed by CHK1 inhibitors in non-small cell lung cancer cells

doi: 10.1016/j.lungcan.2013.09.010

Figure Lengend Snippet: (A) Chemosensitization to PMX by the CHK1 inhibitor, MK-8776, as measured by WST-1 assay. Cells were treated with graded concentrations of PMX for 24 h (T0–T24 h) followed by 24 h treatment with MK-8776 at 500 nM (T24–T48 h). CHK1 inhibition preferentially sensitized cells expressing higher levels of CHK1 (H23, H1437 and H1299) as compared to low CHK1-expressing cells (H1993). At as low as 0.3 μM PMX concentration there was significant reduction in percent of proliferating cells in H23 (p = 0.0012), H1437 (p = 0.0006) and H1299 (p = 0.0006). No significant change in H1993 cells (p = 0.26). Each experiment was performed using 3 replicates for each drug concentration. All experiments were repeated a minimum of 3 times. (B) Chemosensitization by clonogenic survival assay shown in log scale. Low CHK1-expressing H1993 cells are not sensitized to PMX in combination with MK-8776, whereas over-expressing cell lines are sensitized.

Article Snippet: PathScan CHK1 Sandwich ELISA kit (Cell Signaling) was used according to manufacturer’s protocol.

Techniques: WST-1 Assay, Inhibition, Expressing, Concentration Assay, Clonogenic Cell Survival Assay

Journal: Lung cancer (Amsterdam, Netherlands)

Article Title: CHK1 levels correlate with sensitization to pemetrexed by CHK1 inhibitors in non-small cell lung cancer cells

doi: 10.1016/j.lungcan.2013.09.010

Figure Lengend Snippet: (A) Western Blot analyses of the MK-8776 plus PMX in low and high-expressing CHK1 cell lines. The higher CHK1 expressing line, H1299, displays a greater response in S345 autophosphorylation with combination treatment. Also of note, H1299 appears to have a higher baseline CDC25A expression which is reduced with PMX treatment alone. The addition of a CHK1 inhibitor increases CDC25A expression indicating mitotic entry. (B) Western Blot analysis of CHK1 siRNA treatment showing knockout of CHK1 protein expression at 24 and 48 h. In addition, cells were treated with non-target siRNAs (NT) and Lipofectamine RNAiMAX transfection reagent (Mock). (C) Effect of CHK1 siRNA on proliferation in H1993 and H1299 cell lines. H1299 cells show a greater reduction in proliferation than H1993 cells when treated with CHK1 siRNA vs NT siRNA. The combination of CHK1 siRNA and MK-8776 at 750 nM had no effect compared to siRNA use alone.

Article Snippet: PathScan CHK1 Sandwich ELISA kit (Cell Signaling) was used according to manufacturer’s protocol.

Techniques: Western Blot, Expressing, Knock-Out, Transfection

Journal: The American Journal of Pathology

Article Title: Conjugated Bile Acids Promote Invasive Growth of Esophageal Adenocarcinoma Cells and Cancer Stem Cell Expansion via Sphingosine 1-Phosphate Receptor 2–Mediated Yes-Associated Protein Activation

doi: 10.1016/j.ajpath.2018.05.015

Figure Lengend Snippet: List of Antibodies Used in This Study

Article Snippet: Antibodies used in this study are listed in . table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Species Source Catalog number Application (dilution) S1PR1 Rabbit Abcam (Cambridge, UK) Ab23695 WB (1:500) S1PR2 Rabbit Santa Cruz Biotechnology (Dallas, TX) sc-25491 WB (1:200) S1PR3 Rabbit Santa Cruz Biotechnology sc-30024 WB (1:200) E-cadherin Rabbit Santa Cruz Biotechnology sc-7870 WB (1:200)/IF (1:50) Vimentin Rabbit Cell Signaling Technology (Danvers, MA) mAb 5741 WB (1:1000) CTGF Rabbit Sigma-Aldrich (St. Louis, MO) Sab1300909 WB (1:500) β-Catenin Mouse Santa Cruz Biotechnology sc-7963 WB (1:500) YAP Rabbit Sigma-Aldrich Y0771 WB (1:1000) p-YAP Rabbit Sigma-Aldrich Y4645 WB (1:1000) Lamin B Goat Santa Cruz Biotechnology sc-6216 WB (1:500) β-Actin (JLA20) Mouse DSHB at University of Iowa (Iowa City, IA) JLA20 WB (1:500) Open in a separate window CTGF, connective tissue growth factor; DSHB, Developmental Studies Hybridoma Bank; IF, immunofluorescence; mAb, monoclonal antibody; p-YAP, phosphorylated YAP; S1PR, sphingosine 1-phosphate receptor; WB, Western blotting.

Techniques:

Journal: The American Journal of Pathology

Article Title: Conjugated Bile Acids Promote Invasive Growth of Esophageal Adenocarcinoma Cells and Cancer Stem Cell Expansion via Sphingosine 1-Phosphate Receptor 2–Mediated Yes-Associated Protein Activation

doi: 10.1016/j.ajpath.2018.05.015

Figure Lengend Snippet: Sphingosine 1-phosphate receptor 2 (S1PR2) activation promotes transforming growth factor (TGF)-β–induced epithelial-to-mesenchymal transition in esophageal adenocarcinoma cells. A: Relative mRNA levels of E-cadherin in OE-19 and OE-33 cells. B: Representative images and relative protein levels of E-cadherin in OE-19 and OE-33 cells. C: Representative bright-field images and immunofluorescence images against E-cadherin of OE-19 and OE-33 cells. D: OE-19 and OE-33 cells were treated with 2.5 μg/mL TGF-β, 100 μmol/L taurocholate (TCA), or both for 72 hours. Representative bright-field images and immunofluorescence images of E-cadherin are shown. E: Protein levels of E-cadherin and vimentin in total lysate and β-catenin in nuclear and cytosolic fractions in OE-19 and OE-33 cells were determined by Western blots. Relative protein levels were analyzed using Lamin B as loading control for nuclear fraction and β-actin as loading control for cytosolic fraction and total lysate. Data are expressed as means ± SEM (A, B, and E). n = 3 (A, B, and E). ∗∗P < 0.01 versus OE-19; †P < 0.05, ††P < 0.01 versus control; ‡P < 0.05 versus no JTE-013 (S1PR2 antagonist); §P < 0.05 versus TGF-β. CTGF, connective tissue growth factor; E-Cad, E-cadherin.

Article Snippet: Antibodies used in this study are listed in . table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Species Source Catalog number Application (dilution) S1PR1 Rabbit Abcam (Cambridge, UK) Ab23695 WB (1:500) S1PR2 Rabbit Santa Cruz Biotechnology (Dallas, TX) sc-25491 WB (1:200) S1PR3 Rabbit Santa Cruz Biotechnology sc-30024 WB (1:200) E-cadherin Rabbit Santa Cruz Biotechnology sc-7870 WB (1:200)/IF (1:50) Vimentin Rabbit Cell Signaling Technology (Danvers, MA) mAb 5741 WB (1:1000) CTGF Rabbit Sigma-Aldrich (St. Louis, MO) Sab1300909 WB (1:500) β-Catenin Mouse Santa Cruz Biotechnology sc-7963 WB (1:500) YAP Rabbit Sigma-Aldrich Y0771 WB (1:1000) p-YAP Rabbit Sigma-Aldrich Y4645 WB (1:1000) Lamin B Goat Santa Cruz Biotechnology sc-6216 WB (1:500) β-Actin (JLA20) Mouse DSHB at University of Iowa (Iowa City, IA) JLA20 WB (1:500) Open in a separate window CTGF, connective tissue growth factor; DSHB, Developmental Studies Hybridoma Bank; IF, immunofluorescence; mAb, monoclonal antibody; p-YAP, phosphorylated YAP; S1PR, sphingosine 1-phosphate receptor; WB, Western blotting.

Techniques: Activation Assay, Immunofluorescence, Western Blot, Control