Journal: PLoS ONE

Article Title: Recombinant Thrombomodulin Protects Mice against Histone-Induced Lethal Thromboembolism

doi: 10.1371/journal.pone.0075961

Figure Lengend Snippet: Plasma histone H3 levels were significantly higher in non-survivors (n = 16, minimum = 0, maximum = 1464.4, median = 15.5) compared with healthy volunteers (n = 15, minimum = 0, maximum = 0) as analyzed by Steel-Dwass test.

Article Snippet: Washed platelets resuspended in Tyrode-HEPES buffer (pH 7.35) or platelet-rich plasma anticoagulated with either sodium citrate or hirudin were stimulated with recombinant human histone H3 or H4 (New England Biolabs, Ipswich, MA), or with collagen.

Techniques:

Journal: PLoS ONE

Article Title: Recombinant Thrombomodulin Protects Mice against Histone-Induced Lethal Thromboembolism

doi: 10.1371/journal.pone.0075961

Figure Lengend Snippet: (A) Binding assays with a quartz crystal microbalance (QCM) twin sensor system. Two channels of a sensor chip were coated with either histones (1 mg/ml) or BSA (1 mg/ml). The sensor chip was placed into the NAPiCOS Auto and then perfused with rTM (1 mg/ml). The interaction between molecules was recognized as the change in frequency of a quartz crystal resonator. (B) Inhibition of histone H4-mediated platelet aggregation by rTM. Washed platelets were stimulated with histone H4 (5 µg/ml) preincubated with rTM (5-25 µg/ml) or BSA (25 µg/ml). rTM, but not BSA, inhibited histone-induced platelet aggregation (n = 3-7 per group, mean ± S.D.). (C) Inhibition of histone H3-induced platelet aggregation by rTM. Washed platelets were stimulated with histone H3 (25 µg/ml) or collagen (1.44 µg/ml) in the presence or absence of rTM (15 µg/ml). rTM inhibited histone-induced platelet aggregation, but not collagen-induced platelet aggregation. Representative data of three independent experiments are shown. ** P < 0.01 compared with the histone-alone group.

Article Snippet: Washed platelets resuspended in Tyrode-HEPES buffer (pH 7.35) or platelet-rich plasma anticoagulated with either sodium citrate or hirudin were stimulated with recombinant human histone H3 or H4 (New England Biolabs, Ipswich, MA), or with collagen.

Techniques: Binding Assay, Inhibition

Journal: PLoS ONE

Article Title: Nuclear Legumain Activity in Colorectal Cancer

doi: 10.1371/journal.pone.0052980

Figure Lengend Snippet: (A) Immunoblots showing the cleavage of intact (lane 10) recombinant human histone H3.1 in a dose dependent manner by purified mature 36 kDa bovine legumain (bovLeg, lane 1–3) and auto-activated intermediate form (46 kDa) of recombinant human legumain (rhLeg, lane 6–8). The addition of recombinant human cystatin E/M (lane 4 and 8) efficiently blocked legumain activity and resulted in almost complete rescue of histone H3.1 from proteolytic cleavage. Uncut immunoblots . (B) Immunoblot of histone H3.1 showing the dose-dependent production of a 12 kDa cleavage product after incubation of recombinant histone H3.1 with fully mature 36 kDa bovine legumain in a buffer with pH 7.0 (lane 1–3). Addition of recombinant human cystatin E/M efficiently blocked legumain activity and resulted in virtually no formation of the 12 kDa cleavage product (lane 4).

Article Snippet: Bovine legumain was isolated from kidney as described by Yamane et al. . Human recombinant histone H3.1 (New England BioLabs, M2503S) was added to 50 µl assay buffer (50 mM MES, 250 mM NaCl, pH 5.0 or pH 7.0) with or without cystatin E/M and with final addition of either active human or bovine legumain.

Techniques: Western Blot, Recombinant, Purification, Activity Assay, Incubation

Journal: Epigenetics & Chromatin

Article Title: De novo methylation of histone H3K23 by the methyltransferases EHMT1/GLP and EHMT2/G9a

doi: 10.1186/s13072-022-00468-1

Figure Lengend Snippet: Pharmacologic inhibition of EHMT1/GLP and EHMT2/GLP decreases H3K9 and H3K23 methylation in vitro. A A dose titration was performed using the competitive inhibitor UNC0642 with either EHMT1/GLP or EHMT2/G9a, and the enzyme activity in the presence of the inhibitor or DMSO (the vehicle) was measured using the MTase-Glo assay. B Recombinant histone H3.1 was incubated with either EHMT1/GLP or EHMT2/G9a, SAM, and either DMSO or 10 μM UNC0642 for 24 h, and subjected to western blotting to detect loss of various H3K9 and H3K23 methylation states in the in vitro HMT inhibition reactions

Article Snippet: In vitro histone methyltransferase reactions were prepared in buffer containing 20 mM Tris, pH 8.0, 50 mM NaCl, 1 mM EDTA, 3 mM MgCl 2 , 0.1 mg/mL BSA, 1 mM DTT, 20 μM S-adenosyl methionine (Promega, Cat. V7601), 1 μg human recombinant H3.1 (New England Biolabs, Cat. M2503S) and 10 ng of either EHMT1/GLP (Active Motif, Cat. 31920) or G9a/EHMT2 (Active Motif, Cat. 31410) and allowed to incubate for 18–24 h at 25 °C.

Techniques: Inhibition, Methylation, In Vitro, Titration, Activity Assay, Glo Assay, Recombinant, Incubation, Western Blot

Journal: eLife

Article Title: Histone H3 clipping is a novel signature of human neutrophil extracellular traps

doi: 10.7554/eLife.68283

Figure Lengend Snippet:

Article Snippet: Peptide, recombinant protein , Recombinant human histone H3.1 , New England Biolabs , NEB:M2503S , .

Techniques: Purification, Western Blot, Immunofluorescence, Recombinant, Negative Control, Picogreen Assay, Cytotoxicity Assay, Staining, Software

Journal: eLife

Article Title: Histone H3 clipping is a novel signature of human neutrophil extracellular traps

doi: 10.7554/eLife.68283

Figure Lengend Snippet:

Article Snippet: Peptide, recombinant protein , Recombinant human histone H3.1 , New England Biolabs , NEB:M2503S , .

Techniques: Purification, Western Blot, Immunofluorescence, Recombinant, Negative Control, Picogreen Assay, Cytotoxicity Assay, Staining, Software

Journal: Frontiers in Cell and Developmental Biology

Article Title: Increased Aurora B expression reduces substrate phosphorylation and induces chromosomal instability

doi: 10.3389/fcell.2022.1018161

Figure Lengend Snippet: Expression of GFP-Aurora B in HeLa cells causes a reduction in Aurora B kinase activity and an increase in mitotic defects. Cells were analyzed 48 h after infection with retroviruses expressing GFP-Aurora B or GFP alone. (A) Immunoblot showing endogenous pT232 (active) Aurora B is reduced in HeLa cells overexpressing Aurora B. Tubulin is shown as a loading control. (B) Immunoblot showing reduced phosphorylation of the bona fide Aurora B substrate histone H3 Serine 10 in HeLa cells infected with retroviruses expressing GFP-Aurora. Tubulin is shown as a loading control. (C–H) Quantitative immunofluorescence demonstrating that expression of GFP-Aurora B decreases phosphorylation of histone H3 (pH3), the kinetochore component DSN1, and INCENP in HeLa cells. (C,E,G) Representative images of pH3, pDSN1, and pINCENP, respectively, in HeLa cells expressing GFP and GFP-Aurora B. (D,F,H) Quantification of pH3 S10 (D) , pDSN1 S109 (F) , and pINCENP TSS (H) demonstrating that expression of GFP-Aurora B significantly reduces phosphorylation of these Aurora B substrates in HeLa cells. n > 10 cells from each of 3 independent experiments. (I–P) GFP-Aurora B expression increases the incidence of mitotic defects. (I) Representative image of a misaligned chromosome (arrow). (J) Expression of GFP-Aurora B in HeLa cells causes an increase in misaligned chromosomes in metaphase. n > 50 metaphase cells from 3 independent experiments. (K) Representative image of a lagging chromosome (arrow). (L) Expression GFP-Aurora B in HeLa cells causes an increase in lagging chromosomes. n > 250 anaphase and telophase cells from each of 3 independent experiments. (M) Representative image of a multipolar spindle. (N) Expression of GFP-Aurora B in HeLa cells causes an increase in multipolar spindles. n > 250 mitotic cells from each of 3 independent experiments. (O) Expression of GFP-Aurora B increases the percentage of binucleate HeLa cells. n > 250 cells from each of three independent experiments. (P) Expression of GFP-Aurora B in HeLa cells impairs their ability to arrest in mitosis after 16 h of treatment with the microtubule poison colcemid, consistent with a weakened mitotic checkpoint. n > 250 cells from each of 3 independent experiments. * p < 0.05.

Article Snippet: Aurora B-3xFLAG was immunoprecipitated with M2-agarose slurry (Selleck B23102) and incubated in buffer (200 mM Tris-HCl, pH 7.5, 100 mM MgCl 2 , 1.5M NaCl, 100 mM NaF) with 1 mM DTT, 1 μM cold ATP, 5 μCi [γ- 32 P] ATP, 2.5 μg histone H3 (NEB M2506S) and with or without the IN-box of recombinant INCENP (Signal Chem I30-31H) at 30°C for 30 min and resolved by SDS-PAGE. γ- 32 P incorporation was visualized by Typhoon TRIO imager (GE Healthcare).

Techniques: Expressing, Activity Assay, Infection, Western Blot, Immunofluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: Increased Aurora B expression reduces substrate phosphorylation and induces chromosomal instability

doi: 10.3389/fcell.2022.1018161

Figure Lengend Snippet: Increased expression of untagged Aurora B inhibits Aurora B kinase activity and causes resistance to Aurora kinase inhibition. (A) Immunoblot showing ≥2- fold overexpression of untagged Aurora B in MDA-MB-231 cells treated with tetracycline. Despite increased protein levels of Aurora B, pT232 (active) Aurora B is reduced. Tubulin is shown as a loading control. (B–E) Quantitative immunofluorescence showing that increased Aurora B expression results in reduced phosphorylation of the Aurora B substrates histone H3 and KNL1. (B,D) Representative images of pH3 and pKNL1 in MDA-MB-231 cells ± 48 h of tet-inducible expression of Aurora B (C,E) Quantification of B and D. n > 10 cells from each of 3 independent experiments. (F–I) 48 h of tet-induced Aurora B expression induces mitotic defects in MDA-MB-231 cells. (F) Representative image of a misaligned chromosome (arrow). (G) Elevated Aurora B expression increases the incidence of misaligned chromosomes. n > 50 metaphases from 3 independent experiments. (H) Representative image of a lagging chromosome (arrow). (I) Increased Aurora B expression induces lagging chromosomes. n > 100 anaphase and telophases from 3 independent experiments. (J–O) Increased Aurora B expression causes resistance to Aurora B inhibition. (J) Primary ARF −/− MEFs grow significantly better than ARF +/+ MEFS after 16 h of exposure to the Aurora B inhibitor ZM447439. Cell number shown is normalized to the number of ARF +/+ cells 10 days after treatment with DMSO. n = 3 independent experiments. (K) Primary ARF −/− MEFs (red), which overexpress endogenous Aurora B, have significantly improved colony forming ability as compared to ARF +/+ MEFs (blue) after 16 h exposure to ZM447439. n = 3 independent experiments. (L) MDA-MB-231 cells expressing untagged Aurora B in response to tetracycline grow significantly better over the course of 10 days than controls after 16 h exposure to ZM447439. n = 3 independent experiments. (M) MDA-MB-231 cells expressing tet-inducible wild type Aurora B exhibit significantly elevated colony forming ability compared to control MDA-MB-231 cells after exposure to ZM447439. n = 3 independent experiments. (N) MCF7 cells expressing Aurora B–GFP in response to tetracycline grow significantly better over the course of 10 days than MCF7 controls after treatment with ZM447439. n = 3 independent experiments. (O) MCF7 cells expressing Aurora B–GFP form significantly more colonies than control cells after exposure to ZM447439. n = 3 independent experiments. * p < 0.05.

Article Snippet: Aurora B-3xFLAG was immunoprecipitated with M2-agarose slurry (Selleck B23102) and incubated in buffer (200 mM Tris-HCl, pH 7.5, 100 mM MgCl 2 , 1.5M NaCl, 100 mM NaF) with 1 mM DTT, 1 μM cold ATP, 5 μCi [γ- 32 P] ATP, 2.5 μg histone H3 (NEB M2506S) and with or without the IN-box of recombinant INCENP (Signal Chem I30-31H) at 30°C for 30 min and resolved by SDS-PAGE. γ- 32 P incorporation was visualized by Typhoon TRIO imager (GE Healthcare).

Techniques: Expressing, Activity Assay, Inhibition, Western Blot, Over Expression, Immunofluorescence