anti histone h4  (Cell Signaling Technology Inc)

 
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    Name:
    Histone H4 L64C1 Mouse mAb ChIP Formulated
    Description:
    Modulation of chromatin structure plays an important role in the regulation of transcription in eukaryotes The nucleosome made up of DNA wound around eight core histone proteins two each of H2A H2B H3 and H4 is the primary building block of chromatin 1 The amino terminal tails of core histones undergo various post translational modifications including acetylation phosphorylation methylation and ubiquitination 2 5 These modifications occur in response to various stimuli and have a direct effect on the accessibility of chromatin to transcription factors and therefore gene expression 6 In most species histone H2B is primarily acetylated at Lys5 12 15 and 20 4 7 Histone H3 is primarily acetylated at Lys9 14 18 23 27 and 56 Acetylation of H3 at Lys9 appears to have a dominant role in histone deposition and chromatin assembly in some organisms 2 3 Phosphorylation at Ser10 Ser28 and Thr11 of histone H3 is tightly correlated with chromosome condensation during both mitosis and meiosis 8 10 Phosphorylation at Thr3 of histone H3 is highly conserved among many species and is catalyzed by the kinase haspin Immunostaining with phospho specific antibodies in mammalian cells reveals mitotic phosphorylation at Thr3 of H3 in prophase and its dephosphorylation during anaphase 11
    Catalog Number:
    2960
    Price:
    None
    Category:
    Primary Antibodies
    Source:
    Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to the amino-terminal sequence of human histone H4.
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    Structured Review

    Cell Signaling Technology Inc anti histone h4
    Effects of neonatal exposure to sevoflurane and sodium butyrate (NaB) treatment on expression of hippocampal acetylated <t>histones</t> H3 and H4. (A, top) Representative Western blot images of acetyl-H3K9, acetyl-H3K14, and total H3 blots in the hippocampal
    Modulation of chromatin structure plays an important role in the regulation of transcription in eukaryotes The nucleosome made up of DNA wound around eight core histone proteins two each of H2A H2B H3 and H4 is the primary building block of chromatin 1 The amino terminal tails of core histones undergo various post translational modifications including acetylation phosphorylation methylation and ubiquitination 2 5 These modifications occur in response to various stimuli and have a direct effect on the accessibility of chromatin to transcription factors and therefore gene expression 6 In most species histone H2B is primarily acetylated at Lys5 12 15 and 20 4 7 Histone H3 is primarily acetylated at Lys9 14 18 23 27 and 56 Acetylation of H3 at Lys9 appears to have a dominant role in histone deposition and chromatin assembly in some organisms 2 3 Phosphorylation at Ser10 Ser28 and Thr11 of histone H3 is tightly correlated with chromosome condensation during both mitosis and meiosis 8 10 Phosphorylation at Thr3 of histone H3 is highly conserved among many species and is catalyzed by the kinase haspin Immunostaining with phospho specific antibodies in mammalian cells reveals mitotic phosphorylation at Thr3 of H3 in prophase and its dephosphorylation during anaphase 11
    https://www.bioz.com/result/anti histone h4/product/Cell Signaling Technology Inc
    Average 97 stars, based on 9 article reviews
    Price from $9.99 to $1999.99
    anti histone h4 - by Bioz Stars, 2020-09
    97/100 stars

    Images

    1) Product Images from "Role of Histone Acetylation in Long-term Neurobehavioral Effects of Neonatal Exposure to Sevoflurane in Rats"

    Article Title: Role of Histone Acetylation in Long-term Neurobehavioral Effects of Neonatal Exposure to Sevoflurane in Rats

    Journal: Neurobiology of disease

    doi: 10.1016/j.nbd.2016.03.017

    Effects of neonatal exposure to sevoflurane and sodium butyrate (NaB) treatment on expression of hippocampal acetylated histones H3 and H4. (A, top) Representative Western blot images of acetyl-H3K9, acetyl-H3K14, and total H3 blots in the hippocampal
    Figure Legend Snippet: Effects of neonatal exposure to sevoflurane and sodium butyrate (NaB) treatment on expression of hippocampal acetylated histones H3 and H4. (A, top) Representative Western blot images of acetyl-H3K9, acetyl-H3K14, and total H3 blots in the hippocampal

    Techniques Used: Expressing, Western Blot

    2) Product Images from "Two herpesviral noncoding PAN RNAs are functionally homologous but do not associate with common chromatin loci"

    Article Title: Two herpesviral noncoding PAN RNAs are functionally homologous but do not associate with common chromatin loci

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007389

    PAN RNA does not associate with chromatin as assessed by CHART or by cell fractionation. . (D) Subcellular fractionation of lytic BCBL-1 cells does not detect PAN RNA in the chromatin fraction by Northern blot. Western blot for FUS (nucleoplasmic), Histone H4 (chromatin) and GAPDH (cytoplasmic) proteins verifies the purity of the three resulting fractions. Three different salts were exchanged in all fractionation buffers: NaCl, LiCl and NH 4 ]. Data are the average of three biological replicates.
    Figure Legend Snippet: PAN RNA does not associate with chromatin as assessed by CHART or by cell fractionation. . (D) Subcellular fractionation of lytic BCBL-1 cells does not detect PAN RNA in the chromatin fraction by Northern blot. Western blot for FUS (nucleoplasmic), Histone H4 (chromatin) and GAPDH (cytoplasmic) proteins verifies the purity of the three resulting fractions. Three different salts were exchanged in all fractionation buffers: NaCl, LiCl and NH 4 ]. Data are the average of three biological replicates.

    Techniques Used: Cell Fractionation, Fractionation, Northern Blot, Western Blot

    3) Product Images from "Haliotis discus discus Sialic Acid-Binding Lectin Reduces the Oncolytic Vaccinia Virus Induced Toxicity in a Glioblastoma Mouse Model"

    Article Title: Haliotis discus discus Sialic Acid-Binding Lectin Reduces the Oncolytic Vaccinia Virus Induced Toxicity in a Glioblastoma Mouse Model

    Journal: Marine Drugs

    doi: 10.3390/md16050141

    The effect of oncoVV-HddSBL on histone modification. C6 glioblastoma cells were treated with PBS, 5 MOI of oncoVV or oncoVV-HddSBL, and histone H3, H4, H3R8, and H4R3 asymmetric dimethylation levels, as well as the expression of FLAG-tagged HddSBL were analyzed by Western blot.
    Figure Legend Snippet: The effect of oncoVV-HddSBL on histone modification. C6 glioblastoma cells were treated with PBS, 5 MOI of oncoVV or oncoVV-HddSBL, and histone H3, H4, H3R8, and H4R3 asymmetric dimethylation levels, as well as the expression of FLAG-tagged HddSBL were analyzed by Western blot.

    Techniques Used: Modification, Expressing, Western Blot

    4) Product Images from "Basal activity of a PARP1-NuA4 complex varies dramatically across cancer cell lines"

    Article Title: Basal activity of a PARP1-NuA4 complex varies dramatically across cancer cell lines

    Journal: Cell reports

    doi: 10.1016/j.celrep.2014.08.009

    Effects of PARP1 activity on histone acetylation and transcription
    Figure Legend Snippet: Effects of PARP1 activity on histone acetylation and transcription

    Techniques Used: Activity Assay

    5) Product Images from "Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death"

    Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

    Journal: Nature

    doi: 10.1038/s41586-019-1167-6

    Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.
    Figure Legend Snippet: Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.

    Techniques Used:

    Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.
    Figure Legend Snippet: Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.

    Techniques Used: Neutralization, Mouse Assay

    NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.
    Figure Legend Snippet: NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.

    Techniques Used: Derivative Assay, Incubation, Recombinant, Immunofluorescence

    NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j
    Figure Legend Snippet: NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j

    Techniques Used: Derivative Assay, Incubation, Confocal Microscopy, Co-Culture Assay, MANN-WHITNEY, Flow Cytometry, Cytometry, Time-lapse Microscopy, Recombinant

    NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.
    Figure Legend Snippet: NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.

    Techniques Used: Derivative Assay, Lysis

    Membrane-pore-forming activity of histone H4.
    Figure Legend Snippet: Membrane-pore-forming activity of histone H4.

    Techniques Used: Activity Assay

    Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.
    Figure Legend Snippet: Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.

    Techniques Used: Chemotaxis Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Marker, Mouse Assay, Cell Counting, Isolation, Incubation, Recombinant

    Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.
    Figure Legend Snippet: Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.

    Techniques Used: Activity Assay, Incubation, Recombinant, Sequencing, Confocal Microscopy, Microscopy

    6) Product Images from "Chronic Sulforaphane Administration Inhibits Resistance to the mTOR-Inhibitor Everolimus in Bladder Cancer Cells"

    Article Title: Chronic Sulforaphane Administration Inhibits Resistance to the mTOR-Inhibitor Everolimus in Bladder Cancer Cells

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21114026

    Protein profile of cell cycle regulating proteins (Rictor, Raptor, histone acetylation, p19, p27) after short-term exposure to 0.5 nM everolimus (E) or 2.5 µM sulforaphane (S) or 0.5 nM everolimus + 2.5 µM sulforaphane (E + S) in synchronized RT112 tumor cells. Controls (C) received cell culture medium alone. One representative of three separate experiments is shown. Each protein analysis was accompanied by a β-actin loading control. One representative internal control is shown. * indicates significant difference to controls, p ≤ 0.05.
    Figure Legend Snippet: Protein profile of cell cycle regulating proteins (Rictor, Raptor, histone acetylation, p19, p27) after short-term exposure to 0.5 nM everolimus (E) or 2.5 µM sulforaphane (S) or 0.5 nM everolimus + 2.5 µM sulforaphane (E + S) in synchronized RT112 tumor cells. Controls (C) received cell culture medium alone. One representative of three separate experiments is shown. Each protein analysis was accompanied by a β-actin loading control. One representative internal control is shown. * indicates significant difference to controls, p ≤ 0.05.

    Techniques Used: Cell Culture

    Protein profile of cell cycle regulating proteins (Rictor, Raptor, histone acetylation, p19, p27) after the long-term application of 0.5 nM everolimus (E) or 2.5 µM sulforaphane (S) or 0.5 nM everolimus + 2.5 µM sulforaphane (E + S) to synchronized RT112 tumor cells. Controls (C) received cell culture medium alone. One representative of three separate experiments is shown. Each protein analysis was accompanied by a β-actin loading control. One representative internal control is shown. The ratio of protein intensity/β-actin intensity is expressed as percentage of controls, set to 100%. * indicates significant difference to controls, p ≤ 0.05.
    Figure Legend Snippet: Protein profile of cell cycle regulating proteins (Rictor, Raptor, histone acetylation, p19, p27) after the long-term application of 0.5 nM everolimus (E) or 2.5 µM sulforaphane (S) or 0.5 nM everolimus + 2.5 µM sulforaphane (E + S) to synchronized RT112 tumor cells. Controls (C) received cell culture medium alone. One representative of three separate experiments is shown. Each protein analysis was accompanied by a β-actin loading control. One representative internal control is shown. The ratio of protein intensity/β-actin intensity is expressed as percentage of controls, set to 100%. * indicates significant difference to controls, p ≤ 0.05.

    Techniques Used: Cell Culture

    7) Product Images from "Two herpesviral noncoding PAN RNAs are functionally homologous but do not associate with common chromatin loci"

    Article Title: Two herpesviral noncoding PAN RNAs are functionally homologous but do not associate with common chromatin loci

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007389

    PAN RNA does not associate with chromatin as assessed by CHART or by cell fractionation. (A) Distribution of PAN RNA CHART peak loci along all 23 human chromosomes and on the viral chromosome (V). KSHV CHART peaks are closed circles and RRV CHART peaks are open circles. CHART peaks are plotted for the time point at which the enrichment of that genomic locus was at its maximum. Only two overlapping peaks were called in KSHV and RRV CHART datasets and are plotted on a separate line. See S1 Appendix for detailed data. (B) qPCR validation of KSHV PAN RNA binding to genomic loci identified by CHART peak calling. DNA associated with KSHV PAN RNA was isolated from BCBL-1 cells 48 h after lytic induction using CHART oligonucleotide set 1. See S3 Appendix for qPCR primer and amplicon details. Peaks were identified in only the KSHV dataset (KSHV PAN CHART, Peaks 1 and 2), only the RRV dataset (RRV PAN CHART, Peaks 4 and 5) or both datasets (Both PAN CHART, Peak 4). The negative control sites are on the viral genome and lacked any CHART enrichment in either dataset. (C) Overlap of CHART peaks with ENCODE eCLIP (275 datasets; 104 proteins) and ENCODE CHIP (162 proteins) peaks. Percentages represent the fraction of PAN RNA CHART peaks that overlap each ENCODE dataset. None of the datasets overlap more than 15% of the PAN RNA CHART peaks. The intensity of blue shading represents the extent of overlap between the datasets. Detailed data are shown in S2 Appendix . (D) Subcellular fractionation of lytic BCBL-1 cells does not detect PAN RNA in the chromatin fraction by Northern blot. Western blot for FUS (nucleoplasmic), Histone H4 (chromatin) and GAPDH (cytoplasmic) proteins verifies the purity of the three resulting fractions. Three different salts were exchanged in all fractionation buffers: NaCl, LiCl and NH 4 Cl. (E) qPCR RNA analysis of the fractionated samples, plotted as the amount of each transcript in the chromatin relative to the nucleoplasm. Kcnq1ot1 is a control chromatin-associated ncRNA [ 28 , 29 ]. Data are the average of three biological replicates.
    Figure Legend Snippet: PAN RNA does not associate with chromatin as assessed by CHART or by cell fractionation. (A) Distribution of PAN RNA CHART peak loci along all 23 human chromosomes and on the viral chromosome (V). KSHV CHART peaks are closed circles and RRV CHART peaks are open circles. CHART peaks are plotted for the time point at which the enrichment of that genomic locus was at its maximum. Only two overlapping peaks were called in KSHV and RRV CHART datasets and are plotted on a separate line. See S1 Appendix for detailed data. (B) qPCR validation of KSHV PAN RNA binding to genomic loci identified by CHART peak calling. DNA associated with KSHV PAN RNA was isolated from BCBL-1 cells 48 h after lytic induction using CHART oligonucleotide set 1. See S3 Appendix for qPCR primer and amplicon details. Peaks were identified in only the KSHV dataset (KSHV PAN CHART, Peaks 1 and 2), only the RRV dataset (RRV PAN CHART, Peaks 4 and 5) or both datasets (Both PAN CHART, Peak 4). The negative control sites are on the viral genome and lacked any CHART enrichment in either dataset. (C) Overlap of CHART peaks with ENCODE eCLIP (275 datasets; 104 proteins) and ENCODE CHIP (162 proteins) peaks. Percentages represent the fraction of PAN RNA CHART peaks that overlap each ENCODE dataset. None of the datasets overlap more than 15% of the PAN RNA CHART peaks. The intensity of blue shading represents the extent of overlap between the datasets. Detailed data are shown in S2 Appendix . (D) Subcellular fractionation of lytic BCBL-1 cells does not detect PAN RNA in the chromatin fraction by Northern blot. Western blot for FUS (nucleoplasmic), Histone H4 (chromatin) and GAPDH (cytoplasmic) proteins verifies the purity of the three resulting fractions. Three different salts were exchanged in all fractionation buffers: NaCl, LiCl and NH 4 Cl. (E) qPCR RNA analysis of the fractionated samples, plotted as the amount of each transcript in the chromatin relative to the nucleoplasm. Kcnq1ot1 is a control chromatin-associated ncRNA [ 28 , 29 ]. Data are the average of three biological replicates.

    Techniques Used: Cell Fractionation, Real-time Polymerase Chain Reaction, RNA Binding Assay, Isolation, Amplification, Negative Control, Chromatin Immunoprecipitation, Fractionation, Northern Blot, Western Blot

    8) Product Images from "Real-time imaging of histone H4 hyperacetylation in living cells"

    Article Title: Real-time imaging of histone H4 hyperacetylation in living cells

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0902150106

    ( A ) Pseudocolored images of the 480 nm/535 nm emission ratio obtained from a COS7 cell expressing the acetylation indicator during mitosis. ( B ) Time courses of the 480 nm/535 nm emission ratio of Histac (●) and Histac-4KR (○) during mitosis. ( C ) Acetylation of histone H4 at K5 and K8 of asynchronous (A) and nocodazole-treated (N) COS7 cells was analyzed by immunoblotting using antibodies against histone H4 acetylated at K5 and K8. Phosphorylated histone H3 (pS10), a mitotic marker, was analyzed by immunoblotting using an antibody against phosphorylated histone H3. COS7 cells were arrested in mitosis by treatment with 10 μg/mL nocodazole for 12 h.
    Figure Legend Snippet: ( A ) Pseudocolored images of the 480 nm/535 nm emission ratio obtained from a COS7 cell expressing the acetylation indicator during mitosis. ( B ) Time courses of the 480 nm/535 nm emission ratio of Histac (●) and Histac-4KR (○) during mitosis. ( C ) Acetylation of histone H4 at K5 and K8 of asynchronous (A) and nocodazole-treated (N) COS7 cells was analyzed by immunoblotting using antibodies against histone H4 acetylated at K5 and K8. Phosphorylated histone H3 (pS10), a mitotic marker, was analyzed by immunoblotting using an antibody against phosphorylated histone H3. COS7 cells were arrested in mitosis by treatment with 10 μg/mL nocodazole for 12 h.

    Techniques Used: Expressing, Marker

    ( A ) COS7 cells expressing Histac and nontransfected COS7 cells were treated with 1 μM TSA. Immunoblot analyses were performed with antibodies against histone H4 acetylated at Lys-5, 8, 12, and 16. ( B and C ) Pseudocolored images and a time course of the emission ratio in the nucleus of a COS7 cell expressing Histac. TSA at a final concentration of 1 μM or vehicle alone was added to the culture at 0 min. ( D ) After photobleaching of Venus within Histac, the cells were treated with 1 μM TSA for 3 h.
    Figure Legend Snippet: ( A ) COS7 cells expressing Histac and nontransfected COS7 cells were treated with 1 μM TSA. Immunoblot analyses were performed with antibodies against histone H4 acetylated at Lys-5, 8, 12, and 16. ( B and C ) Pseudocolored images and a time course of the emission ratio in the nucleus of a COS7 cell expressing Histac. TSA at a final concentration of 1 μM or vehicle alone was added to the culture at 0 min. ( D ) After photobleaching of Venus within Histac, the cells were treated with 1 μM TSA for 3 h.

    Techniques Used: Expressing, Concentration Assay

    ( A ) Schematic representation of the domain structure of Histac. ( B ) Peptide pull-down assay using nonmodified or acetylated (Ac) histone H4 N-terminal tail peptides (upper panel), or partly acetylated peptides (middle and lower panels). Pull-downs were analyzed by immunoblotting using an antibody against GFP.
    Figure Legend Snippet: ( A ) Schematic representation of the domain structure of Histac. ( B ) Peptide pull-down assay using nonmodified or acetylated (Ac) histone H4 N-terminal tail peptides (upper panel), or partly acetylated peptides (middle and lower panels). Pull-downs were analyzed by immunoblotting using an antibody against GFP.

    Techniques Used: Pull Down Assay

    Immunoblot analysis was performed with antibodies against histone H4 acetylated at Lys-5, 8, 12, and 16. COS7 cells were treated with various concentrations of TSA at 37 °C for 3 h ( A and B ). Emission ratio time courses ( C ) and changes in emission ratios ( D ) of cells expressing Histac treated with TSA. Asterisk indicates P
    Figure Legend Snippet: Immunoblot analysis was performed with antibodies against histone H4 acetylated at Lys-5, 8, 12, and 16. COS7 cells were treated with various concentrations of TSA at 37 °C for 3 h ( A and B ). Emission ratio time courses ( C ) and changes in emission ratios ( D ) of cells expressing Histac treated with TSA. Asterisk indicates P

    Techniques Used: Expressing

    ( A ) The schematic representation shows the domain structure of BRDT. YA is a bromodomain mutant, in which Tyr-65 and 308 of BRDT are replaced with Ala. ( B ) Peptide pull-down assay using nonmodified or acetylated (Ac) histone H4 N-terminal tail peptides. Pull-downs were analyzed by immunoblotting using an antibody against GFP. ( C ) Changes in emission ratio of Histac mutants in response to 1 μM TSA for 3 h.
    Figure Legend Snippet: ( A ) The schematic representation shows the domain structure of BRDT. YA is a bromodomain mutant, in which Tyr-65 and 308 of BRDT are replaced with Ala. ( B ) Peptide pull-down assay using nonmodified or acetylated (Ac) histone H4 N-terminal tail peptides. Pull-downs were analyzed by immunoblotting using an antibody against GFP. ( C ) Changes in emission ratio of Histac mutants in response to 1 μM TSA for 3 h.

    Techniques Used: Mutagenesis, Pull Down Assay

    9) Product Images from "Lysosome-mediated processing of chromatin in senescence"

    Article Title: Lysosome-mediated processing of chromatin in senescence

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201212110

    Senescent cells in vitro and in vivo contain reduced histone content. (A) Immunofluorescent confocal images of histone H3 in control or OIS cells (11 d after activation of ER-RASG12V). Yellow arrows indicate cells with most pronounced SAHF. (B) Quantitative immunofluorescence analysis of histone H3 in RS and OIS cells. Representative of two independent experiments. (C) Progressive loss of core histones in senescent IMR90 cells in replicative senescence (RS). Lysates were normalized by cell number and an equal number of cells was loaded per each lane. Lamin A/C was used as a confirmatory loading control. See Materials and methods for details of time course. (D) Immunohistochemistry for histone H3 in human dermal nevus (n) and adjacent epidermis (e). Right-hand panels are expanded from boxed areas on the left. Note decreased nevus staining for H3 (right bottom) as compared with epidermis (right top). Bars: (left) 100 µm; (right) 50 µm. (E) Immunofluorescence of histone H3 (red) and S100 (melanocytes [green]) in human benign nevus and adjacent epidermis. Bars, 20 µm. (F) Higher magnification of boxed regions in E shows reduced staining for H3 in nevus (bottom panels), compared with epidermal melanocytes (top panels). Bars, 20 µm. (G) Quantitative immunofluorescence of H3 in epidermal and nevus melanocytes. Images were obtained in blue (DAPI), green (S100), and red (H3) channels and then H3 intensity was measured in either S100 + epidermal cells, strictly adjacent to the basal membrane, or in S100 + nevus melanocytes. H3 intensity histograms represent fluorescence intensity distribution, combined from three individual nevi. At least 150 epidermal (50 per nevus) and 300 nevus (100 per nevus) melanocytes were assessed.
    Figure Legend Snippet: Senescent cells in vitro and in vivo contain reduced histone content. (A) Immunofluorescent confocal images of histone H3 in control or OIS cells (11 d after activation of ER-RASG12V). Yellow arrows indicate cells with most pronounced SAHF. (B) Quantitative immunofluorescence analysis of histone H3 in RS and OIS cells. Representative of two independent experiments. (C) Progressive loss of core histones in senescent IMR90 cells in replicative senescence (RS). Lysates were normalized by cell number and an equal number of cells was loaded per each lane. Lamin A/C was used as a confirmatory loading control. See Materials and methods for details of time course. (D) Immunohistochemistry for histone H3 in human dermal nevus (n) and adjacent epidermis (e). Right-hand panels are expanded from boxed areas on the left. Note decreased nevus staining for H3 (right bottom) as compared with epidermis (right top). Bars: (left) 100 µm; (right) 50 µm. (E) Immunofluorescence of histone H3 (red) and S100 (melanocytes [green]) in human benign nevus and adjacent epidermis. Bars, 20 µm. (F) Higher magnification of boxed regions in E shows reduced staining for H3 in nevus (bottom panels), compared with epidermal melanocytes (top panels). Bars, 20 µm. (G) Quantitative immunofluorescence of H3 in epidermal and nevus melanocytes. Images were obtained in blue (DAPI), green (S100), and red (H3) channels and then H3 intensity was measured in either S100 + epidermal cells, strictly adjacent to the basal membrane, or in S100 + nevus melanocytes. H3 intensity histograms represent fluorescence intensity distribution, combined from three individual nevi. At least 150 epidermal (50 per nevus) and 300 nevus (100 per nevus) melanocytes were assessed.

    Techniques Used: In Vitro, In Vivo, Activation Assay, Immunofluorescence, Immunohistochemistry, Staining, Fluorescence

    Cytoplasmic histone is processed by a lysosomal/autophagy pathway. (A) Close juxtaposition of senescence-associated CCF (RS) with p62 nuclear bodies. Bars: 10 µm; (inset) 1 µm. (B) Quantitation of control and OIS cells with CCF overlapping p62 (p62 + ) or not (p62 − ). Mean ± SEM, n = 3. (C) Quantitation of proliferating and RS cells with CCF overlapping p62 (p62 + ) or not (p62 − ) and protein ubiquitination (FK2 + ) or not (FK2 − ). Mean ± SEM, n = 3; P
    Figure Legend Snippet: Cytoplasmic histone is processed by a lysosomal/autophagy pathway. (A) Close juxtaposition of senescence-associated CCF (RS) with p62 nuclear bodies. Bars: 10 µm; (inset) 1 µm. (B) Quantitation of control and OIS cells with CCF overlapping p62 (p62 + ) or not (p62 − ). Mean ± SEM, n = 3. (C) Quantitation of proliferating and RS cells with CCF overlapping p62 (p62 + ) or not (p62 − ) and protein ubiquitination (FK2 + ) or not (FK2 − ). Mean ± SEM, n = 3; P

    Techniques Used: Quantitation Assay

    Cytoplasmic chromatin fragments in senescent cells. (A) Cytoplasmic chromatin fragments (CCFs) in senescent cells are strongly positive for histone H3. Yellow arrow marks CCF. (B) Increased proportion of cells with CCFs in RS or OIS. Mean ± SEM, n = 3; P
    Figure Legend Snippet: Cytoplasmic chromatin fragments in senescent cells. (A) Cytoplasmic chromatin fragments (CCFs) in senescent cells are strongly positive for histone H3. Yellow arrow marks CCF. (B) Increased proportion of cells with CCFs in RS or OIS. Mean ± SEM, n = 3; P

    Techniques Used:

    Senescence-associated loss of histones is V-ATPase dependent. (A) Bafilomycin A1 (BafA1) blocks the loss of nuclear histone H3 content in cells undergoing OIS. BafA1 was added to cells at 50 nM on day 5 after RASG12V induction. Cells were harvested 24 h later, and stained for histone H3. Bars, 10 µm. (B) Quantitative histone H3 immunofluorescence in cells from A. Single representative experiment out of three repeats. (C) RASG12V-induced OIS cells were treated with BafA1 as described in A and then assessed for histones by Western blot. LC3 I/II has been used as a control for BafA1 activity. Lysates from 10,000 cells were loaded per well. (D) BafA1 blocks the accumulation of H3cs.1 in cells undergoing OIS. (E) BafA1 and concanamycin A (Con A) block the accumulation of H3cs.1 in RS cells.
    Figure Legend Snippet: Senescence-associated loss of histones is V-ATPase dependent. (A) Bafilomycin A1 (BafA1) blocks the loss of nuclear histone H3 content in cells undergoing OIS. BafA1 was added to cells at 50 nM on day 5 after RASG12V induction. Cells were harvested 24 h later, and stained for histone H3. Bars, 10 µm. (B) Quantitative histone H3 immunofluorescence in cells from A. Single representative experiment out of three repeats. (C) RASG12V-induced OIS cells were treated with BafA1 as described in A and then assessed for histones by Western blot. LC3 I/II has been used as a control for BafA1 activity. Lysates from 10,000 cells were loaded per well. (D) BafA1 blocks the accumulation of H3cs.1 in cells undergoing OIS. (E) BafA1 and concanamycin A (Con A) block the accumulation of H3cs.1 in RS cells.

    Techniques Used: Staining, Immunofluorescence, Western Blot, Activity Assay, Blocking Assay

    10) Product Images from "Neonatal Isoflurane Exposure Induces Neurocognitive Impairment and Abnormal Hippocampal Histone Acetylation in Mice"

    Article Title: Neonatal Isoflurane Exposure Induces Neurocognitive Impairment and Abnormal Hippocampal Histone Acetylation in Mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0125815

    Repeated neonatal exposures to isoflurane induce H4K12 acetylation dysregulation in the CA1 hippocampal region in response to CFC training. (A) Representative images of western blots showing histone acetylation levels in the CA1 hippocampal region at 15 min,1 h, and 24 h after CFC training. Control mice were not subjected to CFC. (B) Quantification of the immunoblots from mice that received repeated exposure to 30% O 2 -enriched air. * p
    Figure Legend Snippet: Repeated neonatal exposures to isoflurane induce H4K12 acetylation dysregulation in the CA1 hippocampal region in response to CFC training. (A) Representative images of western blots showing histone acetylation levels in the CA1 hippocampal region at 15 min,1 h, and 24 h after CFC training. Control mice were not subjected to CFC. (B) Quantification of the immunoblots from mice that received repeated exposure to 30% O 2 -enriched air. * p

    Techniques Used: Western Blot, Mouse Assay

    TSA injection improved CFC performance and hippocampal histone acetylation in mice that received repeated neonatal exposures to isoflurane. (A) TSA injection improved CFC performance in mice that received repeated neonatal exposures to isoflurane. The freezing times of each group (n = 12 mice per group) during CFC training and testing are shown. * p
    Figure Legend Snippet: TSA injection improved CFC performance and hippocampal histone acetylation in mice that received repeated neonatal exposures to isoflurane. (A) TSA injection improved CFC performance in mice that received repeated neonatal exposures to isoflurane. The freezing times of each group (n = 12 mice per group) during CFC training and testing are shown. * p

    Techniques Used: Injection, Mouse Assay

    11) Product Images from "MicroRNA-mediated mRNA Translation Activation in Quiescent Cells and Oocytes Involves Recruitment of a Nuclear microRNP"

    Article Title: MicroRNA-mediated mRNA Translation Activation in Quiescent Cells and Oocytes Involves Recruitment of a Nuclear microRNP

    Journal: Scientific Reports

    doi: 10.1038/srep00842

    Expression of FXR1-iso-a to overexpress FXR1 levels increases the cytoplasmic levels of AGO2 and rescues translation activation of CX mRNA by miRcxcr4 in asynchronous, low density proliferating cells. (a) Western analysis of nuclear and cytoplasmic extracts from asynchronous, low density proliferating HEK293 cells (slowly proliferating, mostly lag phase cells) that express either GFP control or FXR1-iso-a (FXR1, 1µg transfected/ 1x10 5 cells/ml).FXR1-iso-a overexpression leads to increased FXR1 levels as well as increased cytoplasmic AGO2 levels with some increase in the nucleus (short exp = short exposure of the saturated levels present in the original exposure above). Histone H4 and tubulin served as nuclear and cytoplasmic markers, respectively. (b) Immunoprecipitation of AGO2 from cytoplasmic extracts expressing FXR1-iso-a or GFP in asynchronous, low density proliferating cells demonstrated increased interaction between AGO2 and FXR1-iso-a upon FXR1-iso-a but not GFP expression. (c) Expression of either GFP control or FXR1-iso-a in asynchronous, low density proliferating cells that were subsequently transfected with miRcxcr4 or let-7a (control), CX and Renilla (Ren) reporters, demonstrated rescued upregulated expression of the CX reporter in the presence of miRcxcr4 upon overexpression of FXR1-iso-a but not GFP control. The fold activation depicted (fold activation = the Firefly Luciferase translation value of CX reporter observed in the presence of miRcxcr4 normalized to Renilla over the Luciferase value of CX with control Let-7a microRNA normalized to Renilla that were further normalized to their RNA levels, which do not change significantly as described previously) 54 are compared between samples overexpressing GFP or FXR1-iso-a. The average values of three replicates with standard deviations as error bars are shown.
    Figure Legend Snippet: Expression of FXR1-iso-a to overexpress FXR1 levels increases the cytoplasmic levels of AGO2 and rescues translation activation of CX mRNA by miRcxcr4 in asynchronous, low density proliferating cells. (a) Western analysis of nuclear and cytoplasmic extracts from asynchronous, low density proliferating HEK293 cells (slowly proliferating, mostly lag phase cells) that express either GFP control or FXR1-iso-a (FXR1, 1µg transfected/ 1x10 5 cells/ml).FXR1-iso-a overexpression leads to increased FXR1 levels as well as increased cytoplasmic AGO2 levels with some increase in the nucleus (short exp = short exposure of the saturated levels present in the original exposure above). Histone H4 and tubulin served as nuclear and cytoplasmic markers, respectively. (b) Immunoprecipitation of AGO2 from cytoplasmic extracts expressing FXR1-iso-a or GFP in asynchronous, low density proliferating cells demonstrated increased interaction between AGO2 and FXR1-iso-a upon FXR1-iso-a but not GFP expression. (c) Expression of either GFP control or FXR1-iso-a in asynchronous, low density proliferating cells that were subsequently transfected with miRcxcr4 or let-7a (control), CX and Renilla (Ren) reporters, demonstrated rescued upregulated expression of the CX reporter in the presence of miRcxcr4 upon overexpression of FXR1-iso-a but not GFP control. The fold activation depicted (fold activation = the Firefly Luciferase translation value of CX reporter observed in the presence of miRcxcr4 normalized to Renilla over the Luciferase value of CX with control Let-7a microRNA normalized to Renilla that were further normalized to their RNA levels, which do not change significantly as described previously) 54 are compared between samples overexpressing GFP or FXR1-iso-a. The average values of three replicates with standard deviations as error bars are shown.

    Techniques Used: Expressing, Activation Assay, Western Blot, Transfection, Over Expression, Immunoprecipitation, Luciferase

    Xenopus AGO and an endogenous microRNA, miR16, are substantially present in the nucleus in immature oocytes. All extracts (RNA and protein) and nucleus/cytoplasm isolation were prepared as described 37 38 (Methods). (a) Western analysis of total (T), nuclear (N) and cytoplasmic (C) sonicated extracts from 20 immature, folliculated oocytes using histone H4 as a marker for nuclei and tubulin as a cytoplasmic marker. A lane was left after each fraction loaded to preclude signals from spillover (observed with the tubulin signal in the lane between the cytoplasm and total samples). AGO is more clearly detected when cytoplasmic extracts are sonicated (shown) than in soluble cytoplasmic extracts (all other figures) 38 . (b) The antibody used for AGO analysis, detects expression from Flag-tagged Xenopus AGO and human (hAGO2) clones. In vitro transcription coupled translation RRL extracts (Promega) were used to express control GFP, Flag-tagged Xenopus AGO and human AGO2 (hAGO2) that were further subject to Flag purification and subsequent Western analyses with the AGO2 antibody used (Millipore, AGO2 antibody) and cross-checked with Flag antibody. (c) Northern analysis of endogenous miR16 in nuclei and cytoplasm of immature oocytes using probes against U6 as a nuclear marker and 5.8S rRNA as a cytoplasmic marker. Endogenous miR16 is substantially nuclear. (d) Splint ligation reactions for detection of miR16/B3 5′ trimmed form using a specific B3 or a control bridging oligonucleotide (bridge splint) as described previously 38 with nuclear and cytoplasmic oocyte RNA samples from 20 oocytes each. In vitro synthesized miR16/B3 form was used as a positive control and tRNA as a negative control.
    Figure Legend Snippet: Xenopus AGO and an endogenous microRNA, miR16, are substantially present in the nucleus in immature oocytes. All extracts (RNA and protein) and nucleus/cytoplasm isolation were prepared as described 37 38 (Methods). (a) Western analysis of total (T), nuclear (N) and cytoplasmic (C) sonicated extracts from 20 immature, folliculated oocytes using histone H4 as a marker for nuclei and tubulin as a cytoplasmic marker. A lane was left after each fraction loaded to preclude signals from spillover (observed with the tubulin signal in the lane between the cytoplasm and total samples). AGO is more clearly detected when cytoplasmic extracts are sonicated (shown) than in soluble cytoplasmic extracts (all other figures) 38 . (b) The antibody used for AGO analysis, detects expression from Flag-tagged Xenopus AGO and human (hAGO2) clones. In vitro transcription coupled translation RRL extracts (Promega) were used to express control GFP, Flag-tagged Xenopus AGO and human AGO2 (hAGO2) that were further subject to Flag purification and subsequent Western analyses with the AGO2 antibody used (Millipore, AGO2 antibody) and cross-checked with Flag antibody. (c) Northern analysis of endogenous miR16 in nuclei and cytoplasm of immature oocytes using probes against U6 as a nuclear marker and 5.8S rRNA as a cytoplasmic marker. Endogenous miR16 is substantially nuclear. (d) Splint ligation reactions for detection of miR16/B3 5′ trimmed form using a specific B3 or a control bridging oligonucleotide (bridge splint) as described previously 38 with nuclear and cytoplasmic oocyte RNA samples from 20 oocytes each. In vitro synthesized miR16/B3 form was used as a positive control and tRNA as a negative control.

    Techniques Used: Isolation, Western Blot, Sonication, Marker, Expressing, In Vitro, Purification, Northern Blot, Ligation, Synthesized, Positive Control, Negative Control

    Expression of FXR1-iso-a to overexpress FXR1 levels increases the cytoplasmic levels of AGO and rescues translation activation of CX mRNA by miRcxcr4, injected cytoplasmically into oocytes. (a) Oocytes were nuclear injected with DNA plasmids to express either GFP control or FXR1-iso-a in oocytes and were subsequently cytoplasmically injected with miRcxcr4 or control let-7a, CX and Renilla reporters.Upregulated expression of the CX reporter in the presence of miRcxcr4 was rescued upon overexpression of FXR1-iso-a but not with GFP control or with the control microRNA. (b) Western blot analysis of nuclei (N) and cytoplasm (C) extracts (soluble extracts that were not sonicated) from 20 immature, folliculated oocytes each that were injected with DNA plasmids to express either GFP control or FXR1-iso-a. Histone H4 and actin served as controls. FXR1-iso-a expression leads to increased FXR1 levels as well as increased cytoplasmic levels of AGO. AGO is more clearly detected when cytoplasmic extracts are sonicated ( Fig. 2a ) compared to soluble cytoplasmic extracts (this figure) 38 . (c) mRNA levels of the CX reporter normalized to Renilla reporter levels as analyzed by qRT-PCR with GFP or FXR1-iso-a expression do not correlate with translation changes in (a). (d) AGO2 (Wako) immunoprecipitates from cytoplasmic samples expressing either GFP control or FXR1-iso-a were analyzed for co-immunoprecipitation of FXR1. Increased levels of FXR1 were immunoprecipitated with samples overexpressing FXR1-iso-a. RPA antibody immunoprecipitation served as an antibody control. Increased AGO and FXR1 levels can be observed in the Input lane of the samples overexpressing FXR1-iso-a compared to Actin levels used as a loading control; the AGO antibody amounts (1/3) used for immunoprecipitation are limiting. The average values of three replicates with standard deviations as error bars are shown in a, c.
    Figure Legend Snippet: Expression of FXR1-iso-a to overexpress FXR1 levels increases the cytoplasmic levels of AGO and rescues translation activation of CX mRNA by miRcxcr4, injected cytoplasmically into oocytes. (a) Oocytes were nuclear injected with DNA plasmids to express either GFP control or FXR1-iso-a in oocytes and were subsequently cytoplasmically injected with miRcxcr4 or control let-7a, CX and Renilla reporters.Upregulated expression of the CX reporter in the presence of miRcxcr4 was rescued upon overexpression of FXR1-iso-a but not with GFP control or with the control microRNA. (b) Western blot analysis of nuclei (N) and cytoplasm (C) extracts (soluble extracts that were not sonicated) from 20 immature, folliculated oocytes each that were injected with DNA plasmids to express either GFP control or FXR1-iso-a. Histone H4 and actin served as controls. FXR1-iso-a expression leads to increased FXR1 levels as well as increased cytoplasmic levels of AGO. AGO is more clearly detected when cytoplasmic extracts are sonicated ( Fig. 2a ) compared to soluble cytoplasmic extracts (this figure) 38 . (c) mRNA levels of the CX reporter normalized to Renilla reporter levels as analyzed by qRT-PCR with GFP or FXR1-iso-a expression do not correlate with translation changes in (a). (d) AGO2 (Wako) immunoprecipitates from cytoplasmic samples expressing either GFP control or FXR1-iso-a were analyzed for co-immunoprecipitation of FXR1. Increased levels of FXR1 were immunoprecipitated with samples overexpressing FXR1-iso-a. RPA antibody immunoprecipitation served as an antibody control. Increased AGO and FXR1 levels can be observed in the Input lane of the samples overexpressing FXR1-iso-a compared to Actin levels used as a loading control; the AGO antibody amounts (1/3) used for immunoprecipitation are limiting. The average values of three replicates with standard deviations as error bars are shown in a, c.

    Techniques Used: Expressing, Activation Assay, Injection, Over Expression, Western Blot, Sonication, Quantitative RT-PCR, Immunoprecipitation, Recombinase Polymerase Amplification

    12) Product Images from "Haliotis discus discus Sialic Acid-Binding Lectin Reduces the Oncolytic Vaccinia Virus Induced Toxicity in a Glioblastoma Mouse Model"

    Article Title: Haliotis discus discus Sialic Acid-Binding Lectin Reduces the Oncolytic Vaccinia Virus Induced Toxicity in a Glioblastoma Mouse Model

    Journal: Marine Drugs

    doi: 10.3390/md16050141

    The effect of oncoVV-HddSBL on histone modification. C6 glioblastoma cells were treated with PBS, 5 MOI of oncoVV or oncoVV-HddSBL, and histone H3, H4, H3R8, and H4R3 asymmetric dimethylation levels, as well as the expression of FLAG-tagged HddSBL were analyzed by Western blot.
    Figure Legend Snippet: The effect of oncoVV-HddSBL on histone modification. C6 glioblastoma cells were treated with PBS, 5 MOI of oncoVV or oncoVV-HddSBL, and histone H3, H4, H3R8, and H4R3 asymmetric dimethylation levels, as well as the expression of FLAG-tagged HddSBL were analyzed by Western blot.

    Techniques Used: Modification, Expressing, Western Blot

    13) Product Images from "Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death"

    Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

    Journal: Nature

    doi: 10.1038/s41586-019-1167-6

    Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.
    Figure Legend Snippet: Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.

    Techniques Used:

    Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.
    Figure Legend Snippet: Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.

    Techniques Used: Neutralization, Mouse Assay

    NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.
    Figure Legend Snippet: NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.

    Techniques Used: Derivative Assay, Incubation, Recombinant, Immunofluorescence

    NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j
    Figure Legend Snippet: NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j

    Techniques Used: Derivative Assay, Incubation, Confocal Microscopy, Co-Culture Assay, MANN-WHITNEY, Flow Cytometry, Cytometry, Time-lapse Microscopy, Recombinant

    NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.
    Figure Legend Snippet: NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.

    Techniques Used: Derivative Assay, Lysis

    Membrane-pore-forming activity of histone H4.
    Figure Legend Snippet: Membrane-pore-forming activity of histone H4.

    Techniques Used: Activity Assay

    Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.
    Figure Legend Snippet: Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.

    Techniques Used: Chemotaxis Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Marker, Mouse Assay, Cell Counting, Isolation, Incubation, Recombinant

    Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.
    Figure Legend Snippet: Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.

    Techniques Used: Activity Assay, Incubation, Recombinant, Sequencing, Confocal Microscopy, Microscopy

    14) Product Images from "CstF-64 supports pluripotency and regulates cell cycle progression in embryonic stem cells through histone 3′ end processing"

    Article Title: CstF-64 supports pluripotency and regulates cell cycle progression in embryonic stem cells through histone 3′ end processing

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku551

    CstF-64 is required for S-phase entry and histone expression. ( A ) Cell cycle analysis of WT and Cstf2 E6 ESCs mitotically synchronized (0 h) with nocodazole and subsequently released for 10 h in 2 h time points (2–10 h). Cells were stained with PI and analyzed using flow cytometry. ( B ) Corresponding percentages of synchronized and released WT and Cstf2 E6 ESCs in the cell cycle phases, G 1 (orange), S (blue) and G 2 /M (brown). ( C , D ) Western blot analysis detecting the expression of CstF-64, τCstF-64, CstF-77 and the histone processing components, FLASH and SLBP in mitotically synchronized WT and Cstf2 E6 ESCs that were released for 10 h post block. Cyclin B1 expression indicates G 2 /M phase transition. ( E ) Comparative western blot analysis of the core histone families, H2B, H3 and H4 in mitotically synchronized WT and Cstf2 E6 ESCs.
    Figure Legend Snippet: CstF-64 is required for S-phase entry and histone expression. ( A ) Cell cycle analysis of WT and Cstf2 E6 ESCs mitotically synchronized (0 h) with nocodazole and subsequently released for 10 h in 2 h time points (2–10 h). Cells were stained with PI and analyzed using flow cytometry. ( B ) Corresponding percentages of synchronized and released WT and Cstf2 E6 ESCs in the cell cycle phases, G 1 (orange), S (blue) and G 2 /M (brown). ( C , D ) Western blot analysis detecting the expression of CstF-64, τCstF-64, CstF-77 and the histone processing components, FLASH and SLBP in mitotically synchronized WT and Cstf2 E6 ESCs that were released for 10 h post block. Cyclin B1 expression indicates G 2 /M phase transition. ( E ) Comparative western blot analysis of the core histone families, H2B, H3 and H4 in mitotically synchronized WT and Cstf2 E6 ESCs.

    Techniques Used: Expressing, Cell Cycle Assay, Staining, Flow Cytometry, Cytometry, Western Blot, Blocking Assay, Sublimation

    CstF-64 and τCstF-64 are necessary for normal 3′ end processing of replication-dependent histone mRNAs. ( A ) Western blot analysis of CstF-64 and τCstF-64 expression in WT (lane 1), Cstf2 E6 ESCs transfected with scrambled siRNA (lane 2) and Cstf2 E6 ESCs transfected with siRNA specific for Cstf2t gene (lane 3). ( B ) Relative mRNA expression analysis of polyadenylated and total Hist1h3c histone mRNA in wild type ESCs (orange), Cstf2 E6 cells transfected with scrambled siRNA (blue) or Cstf2 E6 cells transfected with siRNA against Cstf2t gene (brown). ( C ) Western blot analysis of CstF-64 and τCstF-64 expression in WT ESCs either transfected with scrambled siRNA (lane 1) or Cstf2 gene specific siRNA (lane 2). ( D ) Relative mRNA expression analysis of polyadenylated and total Hist1h3c histone mRNA in wild type ESCs transfected with scrambled (orange) or Cstf2 gene specific siRNA (blue). ( E ) Western blot analysis of CstF-64 expression in WT ESCs (lane 1), Cstf2 E6 ESCs (lane 2) and Cstf2 E6 ESCs ectopically expressing CstF-64 (lane 3). ( F ) Corresponding relative mRNA expression analysis of polyadenylated and total Hist1h3c histone mRNA. * denotes P
    Figure Legend Snippet: CstF-64 and τCstF-64 are necessary for normal 3′ end processing of replication-dependent histone mRNAs. ( A ) Western blot analysis of CstF-64 and τCstF-64 expression in WT (lane 1), Cstf2 E6 ESCs transfected with scrambled siRNA (lane 2) and Cstf2 E6 ESCs transfected with siRNA specific for Cstf2t gene (lane 3). ( B ) Relative mRNA expression analysis of polyadenylated and total Hist1h3c histone mRNA in wild type ESCs (orange), Cstf2 E6 cells transfected with scrambled siRNA (blue) or Cstf2 E6 cells transfected with siRNA against Cstf2t gene (brown). ( C ) Western blot analysis of CstF-64 and τCstF-64 expression in WT ESCs either transfected with scrambled siRNA (lane 1) or Cstf2 gene specific siRNA (lane 2). ( D ) Relative mRNA expression analysis of polyadenylated and total Hist1h3c histone mRNA in wild type ESCs transfected with scrambled (orange) or Cstf2 gene specific siRNA (blue). ( E ) Western blot analysis of CstF-64 expression in WT ESCs (lane 1), Cstf2 E6 ESCs (lane 2) and Cstf2 E6 ESCs ectopically expressing CstF-64 (lane 3). ( F ) Corresponding relative mRNA expression analysis of polyadenylated and total Hist1h3c histone mRNA. * denotes P

    Techniques Used: Western Blot, Expressing, Transfection

    Schematic representation of how depletion of CstF-64 increases polyadenylation of replication-dependent histone mRNAs and modulates the cell cycle in ESCs and therefore pluripotency. The panel on the left describes the histone mRNA 3′ end processing complex in wild type ESCs. On the right: modified histone mRNA 3′ end processing complex in the Cstf2 E6 (CstF-64 knockout) cells. Histone mRNA cleavage occurs in wild type mouse ESCs due to interactions of the U7 snRNP with the histone mRNA downstream element (left panel). Other proteins involved in mRNA polyadenylation further associate with the complex, including CstF-64 and symplekin. The complex promotes site-specific cleavage of the histone mRNA by CPSF-73. Together, these processes correlate with normal entry into S-phase and pluripotency. In normal ESCs, a small amount of these cleaved transcripts are polyadenylated. In Cstf2 E6 cells (right panel), CstF-64 is absent, resulting in recruitment of τCstF-64 to the histone 3′ end processing complex (although τCstF-64 interacts more poorly with symplekin). This results in an increase in polyadenylation of the cleaved histone transcripts, presumably by poly(A) polymerase (PAP).
    Figure Legend Snippet: Schematic representation of how depletion of CstF-64 increases polyadenylation of replication-dependent histone mRNAs and modulates the cell cycle in ESCs and therefore pluripotency. The panel on the left describes the histone mRNA 3′ end processing complex in wild type ESCs. On the right: modified histone mRNA 3′ end processing complex in the Cstf2 E6 (CstF-64 knockout) cells. Histone mRNA cleavage occurs in wild type mouse ESCs due to interactions of the U7 snRNP with the histone mRNA downstream element (left panel). Other proteins involved in mRNA polyadenylation further associate with the complex, including CstF-64 and symplekin. The complex promotes site-specific cleavage of the histone mRNA by CPSF-73. Together, these processes correlate with normal entry into S-phase and pluripotency. In normal ESCs, a small amount of these cleaved transcripts are polyadenylated. In Cstf2 E6 cells (right panel), CstF-64 is absent, resulting in recruitment of τCstF-64 to the histone 3′ end processing complex (although τCstF-64 interacts more poorly with symplekin). This results in an increase in polyadenylation of the cleaved histone transcripts, presumably by poly(A) polymerase (PAP).

    Techniques Used: Modification, Knock-Out

    Increase in the polyadenylation of replication-dependent histone mRNAs in Cstf2 E6 cells. ( A ) Fold change of the polyadenylated replication-dependent histones families in the Cstf2 E6 ESCs versus wild type ESCs. Green bars indicate 2-fold or more down-regulation, red bars—2-fold or more up-regulation and black bars no change (
    Figure Legend Snippet: Increase in the polyadenylation of replication-dependent histone mRNAs in Cstf2 E6 cells. ( A ) Fold change of the polyadenylated replication-dependent histones families in the Cstf2 E6 ESCs versus wild type ESCs. Green bars indicate 2-fold or more down-regulation, red bars—2-fold or more up-regulation and black bars no change (

    Techniques Used:

    CstF-64 is a component of the replication-dependent histone mRNA 3′ end-processing complex. ( A ) Western blot analysis of the proteins isolated from a pull-down experiment using anti-U7 snRNP oligonucleotide (lanes 3 and 4) or unrelated mock oligonucleotide (lanes 5 and 6) in WT and Cstf2 E6 ESCs nuclear extracts. 1/100 th of the nuclear extracts from the wild type ESCs (lane 1) and Cstf2 E6 cells (lane 2) before the pull down were also loaded on the gel serving as an input control. Antibodies against the indicated proteins were used. ( B ) Western blot analysis of immunoprecipitation using an anti-CstF-64 antibody in wild type ECCs (lane 3) and Cstf2 E6 (lane 4) cells. 1/100th of the total proteins was also loaded as an input control. Wild type ESCs (lane 1) and Cstf2 E6 cells (lane 2). ( C ) Northern blot of RNA from immunoprecipitation with antibodies against CstF-64 that were hybridized with radiolabeled ribo-probes specific for U7 snRNA or Hist1h3c mRNA. Lane 1, 2 μg of total RNA from wild type ESCs; lane 2, 2 μg of total RNA from Cstf2 E6 cells; lanes 3–4, 2 μg of RNA purified from CstF-64 immunoprecipitation from wild type ESCs (lane 3) or Cstf2 E6 cells (lane 4). ( D ) Western blot analysis of immunoprecipitation with an anti-symplekin antibody in wild type (lane 3) and Cstf2 E6 (lane 4) ESCs protein lysates. IP precipitates from wild type and Cstf2 E6 ESCs were probed for interaction with CstF-64 and τCstF-64 as indicated.
    Figure Legend Snippet: CstF-64 is a component of the replication-dependent histone mRNA 3′ end-processing complex. ( A ) Western blot analysis of the proteins isolated from a pull-down experiment using anti-U7 snRNP oligonucleotide (lanes 3 and 4) or unrelated mock oligonucleotide (lanes 5 and 6) in WT and Cstf2 E6 ESCs nuclear extracts. 1/100 th of the nuclear extracts from the wild type ESCs (lane 1) and Cstf2 E6 cells (lane 2) before the pull down were also loaded on the gel serving as an input control. Antibodies against the indicated proteins were used. ( B ) Western blot analysis of immunoprecipitation using an anti-CstF-64 antibody in wild type ECCs (lane 3) and Cstf2 E6 (lane 4) cells. 1/100th of the total proteins was also loaded as an input control. Wild type ESCs (lane 1) and Cstf2 E6 cells (lane 2). ( C ) Northern blot of RNA from immunoprecipitation with antibodies against CstF-64 that were hybridized with radiolabeled ribo-probes specific for U7 snRNA or Hist1h3c mRNA. Lane 1, 2 μg of total RNA from wild type ESCs; lane 2, 2 μg of total RNA from Cstf2 E6 cells; lanes 3–4, 2 μg of RNA purified from CstF-64 immunoprecipitation from wild type ESCs (lane 3) or Cstf2 E6 cells (lane 4). ( D ) Western blot analysis of immunoprecipitation with an anti-symplekin antibody in wild type (lane 3) and Cstf2 E6 (lane 4) ESCs protein lysates. IP precipitates from wild type and Cstf2 E6 ESCs were probed for interaction with CstF-64 and τCstF-64 as indicated.

    Techniques Used: Western Blot, Isolation, Immunoprecipitation, Northern Blot, Purification

    15) Product Images from "miR-185 mediates lung epithelial cell death after oxidative stress"

    Article Title: miR-185 mediates lung epithelial cell death after oxidative stress

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    doi: 10.1152/ajplung.00392.2015

    Hyperoxia regulates histone acetylation in lung epithelial cells.  A  and  B : Western immunoblot analysis of acetyl-histone H3 ( A ) and acetyl-histone H4 ( B ) protein expressions in human lung epithelial cells (Beas2B) after hyperoxia.  C  and  D : relative acetyl-histone
    Figure Legend Snippet: Hyperoxia regulates histone acetylation in lung epithelial cells. A and B : Western immunoblot analysis of acetyl-histone H3 ( A ) and acetyl-histone H4 ( B ) protein expressions in human lung epithelial cells (Beas2B) after hyperoxia. C and D : relative acetyl-histone

    Techniques Used: Western Blot

    The expression of miR-185 is controlled by HDAC4. A : schematic representation of the genomic location of the human TANGO2 gene. miR-185 locates in the intron of the TANGO2 gene. The acetylation of lysine 27 of the H3 histone protein (H3K27Ac) mark is
    Figure Legend Snippet: The expression of miR-185 is controlled by HDAC4. A : schematic representation of the genomic location of the human TANGO2 gene. miR-185 locates in the intron of the TANGO2 gene. The acetylation of lysine 27 of the H3 histone protein (H3K27Ac) mark is

    Techniques Used: Expressing

    16) Product Images from "Critical role of lysine 134 methylation on histone H2AX for γ-H2AX production and DNA repair"

    Article Title: Critical role of lysine 134 methylation on histone H2AX for γ-H2AX production and DNA repair

    Journal: Nature Communications

    doi: 10.1038/ncomms6691

    SUV39H2 methylates lysine 134 on histone H2AX both in vitro and in vivo . ( a ) The MS/MS spectrum corresponding to the dimethylated histone H2AX peptide (residues 128–142). A 28-Da increase indicates dimethylated Lys 134. Score and Expect show Mascot Ion Score and Expectation value in Mascot Database search results are shown, respectively. ( b ) Validation of K134 methylation on histone H2AX. Recombinant histone H2AX-WT or H2AX-K134A proteins and 3 H-SAM were incubated in the presence of recombinant SUV39H2, and the reaction products were analysed by SDS–PAGE followed by fluorography. The membrane was stained with Ponceau S (lower panel). ( c ) Amino acid sequence alignment of human histone H2A family. Lysine 134 is located in the unique sequence portion of H2AX. ( d ) Amino acid sequence alignment of H2AX unique sequence portion. ( e ) Determination of the titre and specificity of the anti-dimethylated K134 H2AX antibody analysed by enzyme-linked immunosorbent assay. ( f ) Validation of the anti-dimethylated K134 H2AX antibody. Recombinant H2AX-WT or H2AX-K134A proteins and SAM were incubated in the presence or absence of recombinant SUV39H2, and the reaction products were analysed by WB analysis. The intensity of each H2AX K134 dimethylation signal was normalized by the corresponding H2AX. SAHH, S -adenosyl- L -homocysteine hydrolase. Results are the mean±s.d. ( n =3). ( g ) Immunocytochemical analysis of HeLa cells. Cells were stained with an anti-FLAG antibody (green), an anti-H2AX K134me2 antibody (red) and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI (blue)). Non-transfected HeLa cells were used as negative control. Scale bars, 10 μm. ( h ) 293T cells were co-transfected with a FLAG-H2AX-WT or a FLAG-H2AX-K134A, and an empty vector (HA-Mock) or HA-SUV39H2. The samples were immunoblotted with anti-dimethylated K134 H2AX and anti-FLAG antibodies following IP with anti-FLAG. Results are the mean±s.d. ( n =3).
    Figure Legend Snippet: SUV39H2 methylates lysine 134 on histone H2AX both in vitro and in vivo . ( a ) The MS/MS spectrum corresponding to the dimethylated histone H2AX peptide (residues 128–142). A 28-Da increase indicates dimethylated Lys 134. Score and Expect show Mascot Ion Score and Expectation value in Mascot Database search results are shown, respectively. ( b ) Validation of K134 methylation on histone H2AX. Recombinant histone H2AX-WT or H2AX-K134A proteins and 3 H-SAM were incubated in the presence of recombinant SUV39H2, and the reaction products were analysed by SDS–PAGE followed by fluorography. The membrane was stained with Ponceau S (lower panel). ( c ) Amino acid sequence alignment of human histone H2A family. Lysine 134 is located in the unique sequence portion of H2AX. ( d ) Amino acid sequence alignment of H2AX unique sequence portion. ( e ) Determination of the titre and specificity of the anti-dimethylated K134 H2AX antibody analysed by enzyme-linked immunosorbent assay. ( f ) Validation of the anti-dimethylated K134 H2AX antibody. Recombinant H2AX-WT or H2AX-K134A proteins and SAM were incubated in the presence or absence of recombinant SUV39H2, and the reaction products were analysed by WB analysis. The intensity of each H2AX K134 dimethylation signal was normalized by the corresponding H2AX. SAHH, S -adenosyl- L -homocysteine hydrolase. Results are the mean±s.d. ( n =3). ( g ) Immunocytochemical analysis of HeLa cells. Cells were stained with an anti-FLAG antibody (green), an anti-H2AX K134me2 antibody (red) and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI (blue)). Non-transfected HeLa cells were used as negative control. Scale bars, 10 μm. ( h ) 293T cells were co-transfected with a FLAG-H2AX-WT or a FLAG-H2AX-K134A, and an empty vector (HA-Mock) or HA-SUV39H2. The samples were immunoblotted with anti-dimethylated K134 H2AX and anti-FLAG antibodies following IP with anti-FLAG. Results are the mean±s.d. ( n =3).

    Techniques Used: In Vitro, In Vivo, Mass Spectrometry, Methylation, Recombinant, Incubation, SDS Page, Staining, Sequencing, Enzyme-linked Immunosorbent Assay, Western Blot, Transfection, Negative Control, Plasmid Preparation

    SUV39H2 is overexpressed in human lung cancer. ( a ) In vitro methyltransferase analysis of SUV39H2. Recombinant histone H2AX and 3 H-SAM were incubated in the presence or absence of recombinant SUV39H2, and the reaction products were analysed by SDS–PAGE followed by fluorography (upper panel) and stained for total protein(lower panel). ( b ) SUV39H2 mRNA levels in 14 lung cancer cases (NSCLC: 9 cases; SCLC: 5 cases) and 16 normal tissues. ( c ) Quantitative real-time PCR analysis was performed in 14 lung cancer samples and 16 normal tissues (the brain, breast, colon, oesophagus, eye, heart, liver, pancreas, rectum, spleen, stomach, kidney, bladder, testis, placenta and lung) and the result is shown by box-whisker plot. For statistical analysis, Kruskal–Wallis (* P
    Figure Legend Snippet: SUV39H2 is overexpressed in human lung cancer. ( a ) In vitro methyltransferase analysis of SUV39H2. Recombinant histone H2AX and 3 H-SAM were incubated in the presence or absence of recombinant SUV39H2, and the reaction products were analysed by SDS–PAGE followed by fluorography (upper panel) and stained for total protein(lower panel). ( b ) SUV39H2 mRNA levels in 14 lung cancer cases (NSCLC: 9 cases; SCLC: 5 cases) and 16 normal tissues. ( c ) Quantitative real-time PCR analysis was performed in 14 lung cancer samples and 16 normal tissues (the brain, breast, colon, oesophagus, eye, heart, liver, pancreas, rectum, spleen, stomach, kidney, bladder, testis, placenta and lung) and the result is shown by box-whisker plot. For statistical analysis, Kruskal–Wallis (* P

    Techniques Used: In Vitro, Recombinant, Incubation, SDS Page, Staining, Real-time Polymerase Chain Reaction, Whisker Assay

    17) Product Images from "A novel transcriptional signalling pathway mediated by the trafficking protein Ambra1 via scaffolding Atf2 complexes"

    Article Title: A novel transcriptional signalling pathway mediated by the trafficking protein Ambra1 via scaffolding Atf2 complexes

    Journal: bioRxiv

    doi: 10.1101/2020.01.08.899328

    Depletion of Akap8 and Cdk9 reduce p-Atf2 T71 binding to chromatin. (A) SCC FAK-WT cells were transfected with siControl and siAkap8 (siGENOME pool). After 48 h whole cell lysates and chromatin extracts were analysed by Western blot using anti-Ambra1, anti-FAK, anti-p-Atf2 T71, anti-Atf2 and anti-Akap8. Anti-Histone H4 served as a marker for chromatin as well as a loading control. (B) The graph shows relative chromatin protein levels normalised to Histone H4. (C) SCC FAK-WT cells were transfected with siControl, siCdk8 and siCdk9 (siGENOME pool). After 48 h whole cell lysates, nuclear and chromatin extracts were analysed by Western blot using anti-pAtf2 T71, anti-Atf2, anti-Cdk8 and anti-Cdk9. Anti-Lamin A/C, anti-GAPDH and anti-Histone H4 served as a control for the purity of nuclear and chromatin extracts as well as a loading control. (D) The graph shows relative nuclear or chromatin protein levels normalised to Lamin A/C or Histone H4 respectively. (E) Hypothesis model. In the nucleus of SCC cells Ambra1 and Akap8 form a complex and contribute to the recruitment of active Atf2 (p-Atf2 T71) to chromatin, most likely downstream via the Mediator complex component Cdk9. This strongly suggests that Ambra1 might be involved in chromatin remodeling and transcription. Further, together with Akap8 and Atf2 it might co-regulate the expression of a sub-set of genes.
    Figure Legend Snippet: Depletion of Akap8 and Cdk9 reduce p-Atf2 T71 binding to chromatin. (A) SCC FAK-WT cells were transfected with siControl and siAkap8 (siGENOME pool). After 48 h whole cell lysates and chromatin extracts were analysed by Western blot using anti-Ambra1, anti-FAK, anti-p-Atf2 T71, anti-Atf2 and anti-Akap8. Anti-Histone H4 served as a marker for chromatin as well as a loading control. (B) The graph shows relative chromatin protein levels normalised to Histone H4. (C) SCC FAK-WT cells were transfected with siControl, siCdk8 and siCdk9 (siGENOME pool). After 48 h whole cell lysates, nuclear and chromatin extracts were analysed by Western blot using anti-pAtf2 T71, anti-Atf2, anti-Cdk8 and anti-Cdk9. Anti-Lamin A/C, anti-GAPDH and anti-Histone H4 served as a control for the purity of nuclear and chromatin extracts as well as a loading control. (D) The graph shows relative nuclear or chromatin protein levels normalised to Lamin A/C or Histone H4 respectively. (E) Hypothesis model. In the nucleus of SCC cells Ambra1 and Akap8 form a complex and contribute to the recruitment of active Atf2 (p-Atf2 T71) to chromatin, most likely downstream via the Mediator complex component Cdk9. This strongly suggests that Ambra1 might be involved in chromatin remodeling and transcription. Further, together with Akap8 and Atf2 it might co-regulate the expression of a sub-set of genes.

    Techniques Used: Binding Assay, Transfection, Western Blot, Marker, Expressing

    Loss of Ambra1 leads to reduced association of interacting proteins with chromatin. (A)  SCC FAK-WT cells were transfected with siControl and siAmbra1 (siGENOME pool). After 48 h whole cell and nuclear lysates were analysed by Western blot using anti-Ambra1, anti-FAK, anti-Brg1, anti-Akap8, anti-Cdk8, anti-Cdk9, anti-p-Atf2 T71 and anti-Atf2. Anti-Lamin A/C and anti-GAPDH were used as a control for the purity of the nuclear lysates as well as a loading control.  (B)  SCC FAK-WT cells were transfected with siControl and siAmbra1. After 48 h whole cell lysates and chromatin extracts were analysed by Western blot using anti-Ambra1, anti-FAK, anti-Brg1, anti-Akap8, anti-Cdk8, anti-Cdk9, anti-p-Atf2 T71 and anti-Atf2. Anti-Histone H4 served as a marker for chromatin as well as a loading control.  (C)  The graph shows relative chromatin protein levels normalised to Histone H4.
    Figure Legend Snippet: Loss of Ambra1 leads to reduced association of interacting proteins with chromatin. (A) SCC FAK-WT cells were transfected with siControl and siAmbra1 (siGENOME pool). After 48 h whole cell and nuclear lysates were analysed by Western blot using anti-Ambra1, anti-FAK, anti-Brg1, anti-Akap8, anti-Cdk8, anti-Cdk9, anti-p-Atf2 T71 and anti-Atf2. Anti-Lamin A/C and anti-GAPDH were used as a control for the purity of the nuclear lysates as well as a loading control. (B) SCC FAK-WT cells were transfected with siControl and siAmbra1. After 48 h whole cell lysates and chromatin extracts were analysed by Western blot using anti-Ambra1, anti-FAK, anti-Brg1, anti-Akap8, anti-Cdk8, anti-Cdk9, anti-p-Atf2 T71 and anti-Atf2. Anti-Histone H4 served as a marker for chromatin as well as a loading control. (C) The graph shows relative chromatin protein levels normalised to Histone H4.

    Techniques Used: Transfection, Western Blot, Marker

    18) Product Images from "GAPDH Mediates Nitrosylation of Nuclear Proteins"

    Article Title: GAPDH Mediates Nitrosylation of Nuclear Proteins

    Journal: Nature cell biology

    doi: 10.1038/ncb2114

    SNO-GAPDH mediates inhibition of SIRT1 enzymatic activity by nitric oxide ( a ) GSNO treatment inhibits SIRT1 catalytic activity in an in vitro histone deacetylation assay. SIRT1 was pre-treated with the indicated concentration of GSH or GSNO and desalted prior to the assay. ( b ) Pre-incubation with SNO-GAPDH inhibits SIRT1 catalytic activity in vitro . This effect is lost with mutation of GAPDH-T152. The assay was performed as in a . * P
    Figure Legend Snippet: SNO-GAPDH mediates inhibition of SIRT1 enzymatic activity by nitric oxide ( a ) GSNO treatment inhibits SIRT1 catalytic activity in an in vitro histone deacetylation assay. SIRT1 was pre-treated with the indicated concentration of GSH or GSNO and desalted prior to the assay. ( b ) Pre-incubation with SNO-GAPDH inhibits SIRT1 catalytic activity in vitro . This effect is lost with mutation of GAPDH-T152. The assay was performed as in a . * P

    Techniques Used: Inhibition, Activity Assay, In Vitro, Concentration Assay, Incubation, Mutagenesis

    19) Product Images from "Computational analysis of transcriptome signature repurposes low dose trifluoperazine for the treatment of fragile X syndrome in mouse model"

    Article Title: Computational analysis of transcriptome signature repurposes low dose trifluoperazine for the treatment of fragile X syndrome in mouse model

    Journal: bioRxiv

    doi: 10.1101/683169

    Trifluoperazine suppresses the PI3K-Akt-S6K1 signaling cascade but does not affect histone acetylation in hippocampal neurons. ( a to c ) Hippocampal neurons cultured from WT mice were treated with vorinostat (20 μM) ( a ), trichostatin A (20 μM) ( b ), and trifluoperazine (20 μM) ( c ) for 1 hour, and then harvested for Western blot analysis. Level of the acetylated histone H3 and H4 (Ac-H3 and Ac-H4) in vehicle- and drug-treated sample was normalized to total H3 and H4, respectively. ( d ) PI3 kinase activity in lysates from primary WT hippocampal neurons treated with 20 μM trifluoperazine (TFP) or vehicle was determined by ELISA, which measures the concentration of PIP3 converted from PIP2. ( e and f ) Primary WT hippocampal neurons were treated with trifluoperazine (TFP) (20 μM) or LY-294002 (20 μM) for 1 h. Samples harvested immediately after drug or vehicle treatment were analyzed by Western blot for the level of pAkt (normalized to total Akt) ( e ) and pS6K1 (normalized to total S6K1) ( f ). The relative protein level in the vehicle-treated control group was defined as 1 ( a2 , b2 , c2 , e2 , and f2 ). P value was determined by student’s t-test ( a to d ) or one-way ANOVA and post hoc Tukey’s test ( e and f ). *: p
    Figure Legend Snippet: Trifluoperazine suppresses the PI3K-Akt-S6K1 signaling cascade but does not affect histone acetylation in hippocampal neurons. ( a to c ) Hippocampal neurons cultured from WT mice were treated with vorinostat (20 μM) ( a ), trichostatin A (20 μM) ( b ), and trifluoperazine (20 μM) ( c ) for 1 hour, and then harvested for Western blot analysis. Level of the acetylated histone H3 and H4 (Ac-H3 and Ac-H4) in vehicle- and drug-treated sample was normalized to total H3 and H4, respectively. ( d ) PI3 kinase activity in lysates from primary WT hippocampal neurons treated with 20 μM trifluoperazine (TFP) or vehicle was determined by ELISA, which measures the concentration of PIP3 converted from PIP2. ( e and f ) Primary WT hippocampal neurons were treated with trifluoperazine (TFP) (20 μM) or LY-294002 (20 μM) for 1 h. Samples harvested immediately after drug or vehicle treatment were analyzed by Western blot for the level of pAkt (normalized to total Akt) ( e ) and pS6K1 (normalized to total S6K1) ( f ). The relative protein level in the vehicle-treated control group was defined as 1 ( a2 , b2 , c2 , e2 , and f2 ). P value was determined by student’s t-test ( a to d ) or one-way ANOVA and post hoc Tukey’s test ( e and f ). *: p

    Techniques Used: Cell Culture, Mouse Assay, Western Blot, Activity Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay

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    Article Snippet: .. Cell and tissue lysates were analyzed by Western blotting using anti-acetyl histone H3, anti-acetyl histone H4 (Cell Signaling, Beverly, MA, USA), and β-actin (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) antibodies respectively. .. All Western blots for each protein were used with three rabbit tissues and repeated at least two times.

    Incubation:

    Article Title: Defective DNA repair and chromatin organization in patients with quiescent systemic lupus erythematosus
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    Cell Signaling Technology Inc anti phospho histone h2ax ser139 rabbit polyclonal antibody
    PARP-1 inhibitor induces site-specific phosphorylation of <t>H2AX</t> and changes in p53 expression in human breast cancer cells. WCLs prepared from untreated control MCF-7 and BT-20 cells and cells exposed to either one inhibitor alone or both inhibitors in tandem were separated on SDS slab gels (10 or 15%) and analyzed by immunoblotting using indicated antibodies as described in detail in Fig. 1 . The intensity of protein bands representing <t>P-Ser139</t> H2AX and total H2AX protein in each lane was normalized against actin. Then P-Ser139 H2AX/H2AX ratio was calculated and normalized against the ratio calculated for the controls. The relative phosphorylation of H2AX was plotted in the diagram.
    Anti Phospho Histone H2ax Ser139 Rabbit Polyclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc mouse anti histone h3
    Dexmedetomidine increases TMPRSS2 expressions through the α2-adrenergic receptor/STAT3 pathway in the cultured MCF-7. MCF-7 was treated with 1 µM of dexmedetomidine (DEX) for the indicated time periods. (A) Total cellular protein was extracted, and Western blot assay was performed to detect the expression of α2-adrenergic receptor (α2-AdR). (B) Indirect immunofluorescence confocal microscopy was used to image TMPRSS2 (green color), α2-adrenergic receptor (red color), and the nuclei (blue color). A high-power view of the selected area was obtained. Magnification ×63 for all panels. (C) The nuclear fractions were extracted, and Western blot assay was performed for phospho-STAT3 Tyr705 and total STAT3. The nuclear protein <t>histone</t> H3 was used as the loading control. (D) MCF-7 was pretreated for 1 h with different concentrations of the STAT3 inhibitor (WP1066) followed by incubation for 48 h with 1 µM of DEX. The nuclear fractions were extracted, and Western blot assay was performed to validate the inhibition of STAT3 signaling. (E) MCF-7 was pretreated for 1 h with 10 µM of WP1066 followed by incubation for 48 h with 1 µM of DEX. Total cellular protein was extracted, and Western blot assay was performed to detect the expressions of full-length (arrowhead) and the cleaved protease domain (arrow) of TMPRSS2. Data are presented as the mean ± SD. The experiment was conducted independently in triplicate. *, P
    Mouse Anti Histone H3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 89/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit polyclonal anti phospho histone h3
    Plexin-B1/Plexin-B2 suppress epidermal stem cell proliferation during embryonic development. a , Confocal images of immunostainings of murine skin at embryonic day 15.5 (E15.5), 16.5 (E16.5), 18.5 (E18.5) and at an adult stage using anti-phospho-histone H3 (pHH3; red) and anti-keratin 14 (K14; green) antibodies. Dashed lines indicate the basement membrane. Scale bar, 25 μm.  b , Quantification of pHH3-positive cells at different time points of embryonic (E) development and in the adult (mean ± s.e.m.;  n =3 mice per time point).  c , mRNA expression levels of genes encoding B-plexins ( plxnb ) in the murine skin at the indicated time points as determined by quantitative RT-PCR (mean ± s.e.m.;  n =3 mice per time point).  d , Confocal images of immunostainings of murine epidermis at embryonic day 16.5 (E16.5) using anti-Plexin-B1 (green) and anti-Plexin-B2 antibodies (red). Dashed lines indicate the basement membrane. Scale bar, 10 μm.  e , Confocal images of immunostainings of murine epidermis at E16.5 using anti-Plexin-B2 (red), and anti-keratin 14 (K14) or anti-keratin 10 (K10) antibodies (green). Scale bar, 10 μm.  f , Confocal images of immunostainings of murine epidermis at embryonic day 15.5 and 16.5 using an anti-Ki67 (red) and anti-keratin 14 (green) antibodies. “contr.”: control mice (genotype  plxnb1 flox/flox ; plxnb2 flox/flox ), “PlexDKO”: epidermisspecific Plexin-B1/Plexin-B2 double-knockout mice (genotype K14-Cre; plxnb1 flox/flox ; plxnb2 flox/flox ). Blue: DAPI. Scale bar, 50 μm.  g , H  E stained histological sections of murine epidermis at the indicated time points of embryonic development. Brackets indicate epidermal thickness. Scale bar, 50 μm.  h , Quantification of the data in (g) (mean ± s.e.m.; E15.5 and E16.5:  n =7 mice per genotype and time point; unpaired t-test).  i , Confocal images of immunostainings of murine embryonic epidermis using anti-K14 (green) and anti-K10 (red) antibodies. Scale bar, 25 μm.  j , Quantification of K14-positive cells (mean ± s.e.m.;  n =7 mice per genotype and time point; unpaired t-test).
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    Evaluation of γ-Η2ΑΧ foci in irradiated murine epithelial cells, endothelial cells and wild-type fibroblasts. ( A ) Representative panels of immunofluorescence visualization of <t>γ-H2AX</t> foci (green) in murine lung epithelial cells at 200× magnification. Cells were pre-treated with SDG for 6 h and irradiated with γ-rays (2 Gy); ( B – D ) Evaluation of DNA damage by γ-Η2ΑΧ foci counting on ( B ) Epithelial Cells, ( C ) Endothelial Cells, and ( D ) Fibroblasts; ( E – G ) Flow cytometric (FACS) analysis of γ-H2AX foci in irradiated murine lung cells ( E ) Epithelial Cells, ( F ) Endothelial Cells, and ( G ) Fibroblasts. Cells were treated with SDG (50 μM) for 6 h and gamma-irradiated (2 Gy). At desired time interval, cells were processed for FACS analysis. Data were quantified using Summit software and is represented as mean ± SEM. * indicates p
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    PARP-1 inhibitor induces site-specific phosphorylation of H2AX and changes in p53 expression in human breast cancer cells. WCLs prepared from untreated control MCF-7 and BT-20 cells and cells exposed to either one inhibitor alone or both inhibitors in tandem were separated on SDS slab gels (10 or 15%) and analyzed by immunoblotting using indicated antibodies as described in detail in Fig. 1 . The intensity of protein bands representing P-Ser139 H2AX and total H2AX protein in each lane was normalized against actin. Then P-Ser139 H2AX/H2AX ratio was calculated and normalized against the ratio calculated for the controls. The relative phosphorylation of H2AX was plotted in the diagram.

    Journal: Biochemical Pharmacology

    Article Title: PARP inhibition potentiates the cytotoxic activity of C-1305, a selective inhibitor of topoisomerase II, in human BRCA1-positive breast cancer cells

    doi: 10.1016/j.bcp.2012.07.024

    Figure Lengend Snippet: PARP-1 inhibitor induces site-specific phosphorylation of H2AX and changes in p53 expression in human breast cancer cells. WCLs prepared from untreated control MCF-7 and BT-20 cells and cells exposed to either one inhibitor alone or both inhibitors in tandem were separated on SDS slab gels (10 or 15%) and analyzed by immunoblotting using indicated antibodies as described in detail in Fig. 1 . The intensity of protein bands representing P-Ser139 H2AX and total H2AX protein in each lane was normalized against actin. Then P-Ser139 H2AX/H2AX ratio was calculated and normalized against the ratio calculated for the controls. The relative phosphorylation of H2AX was plotted in the diagram.

    Article Snippet: 2.3 Antibodies The following specific primary antibodies were used to detect the relevant proteins: mouse monoclonal anti-p53 antibody DO-1 and rabbit polyclonal anti-H2AX antibody (from BioLegend, San Diego, CA), anti-BRCA-1 rabbit polyclonal antibody (from Upstate Cell Signaling Solutions, Lake Placid, NY), anti-ER-α rabbit polyclonal antibody (from Sigma–Aldrich, St. Louis, MO), anti-phospho-histone H2AX (Ser139) rabbit polyclonal antibody (from Cell Signaling Technology Inc., Danvers, MA), anti-DBC-1 mouse monoclonal antibody (from Abcam plc, Cambridge, UK), and mouse monoclonal anti-actin antibody (clone C4, ICN Biochemicals, Aurora, OH).

    Techniques: Expressing

    Dexmedetomidine increases TMPRSS2 expressions through the α2-adrenergic receptor/STAT3 pathway in the cultured MCF-7. MCF-7 was treated with 1 µM of dexmedetomidine (DEX) for the indicated time periods. (A) Total cellular protein was extracted, and Western blot assay was performed to detect the expression of α2-adrenergic receptor (α2-AdR). (B) Indirect immunofluorescence confocal microscopy was used to image TMPRSS2 (green color), α2-adrenergic receptor (red color), and the nuclei (blue color). A high-power view of the selected area was obtained. Magnification ×63 for all panels. (C) The nuclear fractions were extracted, and Western blot assay was performed for phospho-STAT3 Tyr705 and total STAT3. The nuclear protein histone H3 was used as the loading control. (D) MCF-7 was pretreated for 1 h with different concentrations of the STAT3 inhibitor (WP1066) followed by incubation for 48 h with 1 µM of DEX. The nuclear fractions were extracted, and Western blot assay was performed to validate the inhibition of STAT3 signaling. (E) MCF-7 was pretreated for 1 h with 10 µM of WP1066 followed by incubation for 48 h with 1 µM of DEX. Total cellular protein was extracted, and Western blot assay was performed to detect the expressions of full-length (arrowhead) and the cleaved protease domain (arrow) of TMPRSS2. Data are presented as the mean ± SD. The experiment was conducted independently in triplicate. *, P

    Journal: Annals of Translational Medicine

    Article Title: Dexmedetomidine promotes breast cancer cell migration through Rab11-mediated secretion of exosomal TMPRSS2

    doi: 10.21037/atm.2020.04.28

    Figure Lengend Snippet: Dexmedetomidine increases TMPRSS2 expressions through the α2-adrenergic receptor/STAT3 pathway in the cultured MCF-7. MCF-7 was treated with 1 µM of dexmedetomidine (DEX) for the indicated time periods. (A) Total cellular protein was extracted, and Western blot assay was performed to detect the expression of α2-adrenergic receptor (α2-AdR). (B) Indirect immunofluorescence confocal microscopy was used to image TMPRSS2 (green color), α2-adrenergic receptor (red color), and the nuclei (blue color). A high-power view of the selected area was obtained. Magnification ×63 for all panels. (C) The nuclear fractions were extracted, and Western blot assay was performed for phospho-STAT3 Tyr705 and total STAT3. The nuclear protein histone H3 was used as the loading control. (D) MCF-7 was pretreated for 1 h with different concentrations of the STAT3 inhibitor (WP1066) followed by incubation for 48 h with 1 µM of DEX. The nuclear fractions were extracted, and Western blot assay was performed to validate the inhibition of STAT3 signaling. (E) MCF-7 was pretreated for 1 h with 10 µM of WP1066 followed by incubation for 48 h with 1 µM of DEX. Total cellular protein was extracted, and Western blot assay was performed to detect the expressions of full-length (arrowhead) and the cleaved protease domain (arrow) of TMPRSS2. Data are presented as the mean ± SD. The experiment was conducted independently in triplicate. *, P

    Article Snippet: The following commercial primary antibodies were obtained: rabbit monoclonal anti-TMPRSS2, rabbit anti-heat shock protein 70 (Hsp70), rabbit anti-a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), rabbit anti-EEA1, rabbit anti-collagen IV, rabbit anti-matrix metallopeptidase 16 (MMP16) and rabbit anti-tenascin C antibodies from Abcam (Cambridge, MA, USA); rabbit anti-α2-adrenergic receptor and mouse anti-Hsp90 antibodies from Sigma-Aldrich (St. Louis, MO, USA); mouse anti-signal transducer and activator of transcription 3 (STAT3), mouse anti-GAPDH, mouse anti-histone H3, rabbit anti-Rab35, rabbit anti-fibronectin, rabbit anti-phospho-STAT3Tyr705 , rabbit anti-Rab7, rabbit anti-Rab4 and rabbit anti-Rab11 antibodies from Cell Signaling (Danvers, MA, USA); mouse anti-Rab11 antibody from BD Biosciences (San Jose, CA, USA).

    Techniques: Cell Culture, Western Blot, Expressing, Immunofluorescence, Confocal Microscopy, Incubation, Inhibition

    Plexin-B1/Plexin-B2 suppress epidermal stem cell proliferation during embryonic development. a , Confocal images of immunostainings of murine skin at embryonic day 15.5 (E15.5), 16.5 (E16.5), 18.5 (E18.5) and at an adult stage using anti-phospho-histone H3 (pHH3; red) and anti-keratin 14 (K14; green) antibodies. Dashed lines indicate the basement membrane. Scale bar, 25 μm.  b , Quantification of pHH3-positive cells at different time points of embryonic (E) development and in the adult (mean ± s.e.m.;  n =3 mice per time point).  c , mRNA expression levels of genes encoding B-plexins ( plxnb ) in the murine skin at the indicated time points as determined by quantitative RT-PCR (mean ± s.e.m.;  n =3 mice per time point).  d , Confocal images of immunostainings of murine epidermis at embryonic day 16.5 (E16.5) using anti-Plexin-B1 (green) and anti-Plexin-B2 antibodies (red). Dashed lines indicate the basement membrane. Scale bar, 10 μm.  e , Confocal images of immunostainings of murine epidermis at E16.5 using anti-Plexin-B2 (red), and anti-keratin 14 (K14) or anti-keratin 10 (K10) antibodies (green). Scale bar, 10 μm.  f , Confocal images of immunostainings of murine epidermis at embryonic day 15.5 and 16.5 using an anti-Ki67 (red) and anti-keratin 14 (green) antibodies. “contr.”: control mice (genotype  plxnb1 flox/flox ; plxnb2 flox/flox ), “PlexDKO”: epidermisspecific Plexin-B1/Plexin-B2 double-knockout mice (genotype K14-Cre; plxnb1 flox/flox ; plxnb2 flox/flox ). Blue: DAPI. Scale bar, 50 μm.  g , H  E stained histological sections of murine epidermis at the indicated time points of embryonic development. Brackets indicate epidermal thickness. Scale bar, 50 μm.  h , Quantification of the data in (g) (mean ± s.e.m.; E15.5 and E16.5:  n =7 mice per genotype and time point; unpaired t-test).  i , Confocal images of immunostainings of murine embryonic epidermis using anti-K14 (green) and anti-K10 (red) antibodies. Scale bar, 25 μm.  j , Quantification of K14-positive cells (mean ± s.e.m.;  n =7 mice per genotype and time point; unpaired t-test).

    Journal: bioRxiv

    Article Title: Mechanochemical control of epidermal stem cell divisions by B-plexins

    doi: 10.1101/2020.04.30.070359

    Figure Lengend Snippet: Plexin-B1/Plexin-B2 suppress epidermal stem cell proliferation during embryonic development. a , Confocal images of immunostainings of murine skin at embryonic day 15.5 (E15.5), 16.5 (E16.5), 18.5 (E18.5) and at an adult stage using anti-phospho-histone H3 (pHH3; red) and anti-keratin 14 (K14; green) antibodies. Dashed lines indicate the basement membrane. Scale bar, 25 μm. b , Quantification of pHH3-positive cells at different time points of embryonic (E) development and in the adult (mean ± s.e.m.; n =3 mice per time point). c , mRNA expression levels of genes encoding B-plexins ( plxnb ) in the murine skin at the indicated time points as determined by quantitative RT-PCR (mean ± s.e.m.; n =3 mice per time point). d , Confocal images of immunostainings of murine epidermis at embryonic day 16.5 (E16.5) using anti-Plexin-B1 (green) and anti-Plexin-B2 antibodies (red). Dashed lines indicate the basement membrane. Scale bar, 10 μm. e , Confocal images of immunostainings of murine epidermis at E16.5 using anti-Plexin-B2 (red), and anti-keratin 14 (K14) or anti-keratin 10 (K10) antibodies (green). Scale bar, 10 μm. f , Confocal images of immunostainings of murine epidermis at embryonic day 15.5 and 16.5 using an anti-Ki67 (red) and anti-keratin 14 (green) antibodies. “contr.”: control mice (genotype plxnb1 flox/flox ; plxnb2 flox/flox ), “PlexDKO”: epidermisspecific Plexin-B1/Plexin-B2 double-knockout mice (genotype K14-Cre; plxnb1 flox/flox ; plxnb2 flox/flox ). Blue: DAPI. Scale bar, 50 μm. g , H E stained histological sections of murine epidermis at the indicated time points of embryonic development. Brackets indicate epidermal thickness. Scale bar, 50 μm. h , Quantification of the data in (g) (mean ± s.e.m.; E15.5 and E16.5: n =7 mice per genotype and time point; unpaired t-test). i , Confocal images of immunostainings of murine embryonic epidermis using anti-K14 (green) and anti-K10 (red) antibodies. Scale bar, 25 μm. j , Quantification of K14-positive cells (mean ± s.e.m.; n =7 mice per genotype and time point; unpaired t-test).

    Article Snippet: Antibodies The following primary antibodies were used: guinea pig polyclonal anti-keratin 14 (1:200, Progen, cat. no. GP-CK14), rabbit polyclonal anti-keratin 14 (1:200, Biolegend, cat. no. #905301), rabbit polyclonal anti-keratin 10 (1:200, Biolegend, cat. no. #905401), rabbit polyclonal anti-Ki67 (1:200, Abcam, cat. no. ab15580), rabbit polyclonal anti-phospho-histone H3 (1:200, Cell Signaling, cat. no. #9701), armenian hamster monoclonal anti-Plexin-B2 (for immunostainings; 1:200, eBioscience, cat. #14-5665-85), sheep polyclonal anti-Plexin-B2 (for Western Blot; 1:500, R & D Systems, cat. no. AF6836), rabbit monoclonal anti-E-cadherin (1:200, Cell Signaling, cat. no. #3195), rabbit polyclonal anti-α-catenin (1:200, Invitrogen, cat. no. 71-1200), anti-α-catenin a-18 (for immunostainings on elastomers; 1:10000, ), rabbit monoclonal anti-cleaved Notch1 (NICD, 1:100, Cell Signaling, cat. no. #4147), rabbit monoclonal anti-Yap antibody (1:200, Cell Signaling, cat. no. #14074), mouse monoclonal anti-Yap (for immunostainings on elastomers; 1:300, Santa Cruz; sc-101199), rabbit monoclonal anti-active Yap (1:200, Abcam, cat. no. ab205270), rabbit polyclonal anti-phospho-myosin light chain 2 (Ser19) (1:200, Cell Signaling, cat. no. #3671), rabbit polyclonal anti-phospho-myosin light chain 2 (Thr18/Ser19) (for immunostainings on elastomers; 1:200, Cell Signaling; cat. no. #3674).

    Techniques: Mouse Assay, Expressing, Quantitative RT-PCR, Double Knockout, Staining

    Evaluation of γ-Η2ΑΧ foci in irradiated murine epithelial cells, endothelial cells and wild-type fibroblasts. ( A ) Representative panels of immunofluorescence visualization of γ-H2AX foci (green) in murine lung epithelial cells at 200× magnification. Cells were pre-treated with SDG for 6 h and irradiated with γ-rays (2 Gy); ( B – D ) Evaluation of DNA damage by γ-Η2ΑΧ foci counting on ( B ) Epithelial Cells, ( C ) Endothelial Cells, and ( D ) Fibroblasts; ( E – G ) Flow cytometric (FACS) analysis of γ-H2AX foci in irradiated murine lung cells ( E ) Epithelial Cells, ( F ) Endothelial Cells, and ( G ) Fibroblasts. Cells were treated with SDG (50 μM) for 6 h and gamma-irradiated (2 Gy). At desired time interval, cells were processed for FACS analysis. Data were quantified using Summit software and is represented as mean ± SEM. * indicates p

    Journal: International Journal of Molecular Sciences

    Article Title: The Flaxseed-Derived Lignan Phenolic Secoisolariciresinol Diglucoside (SDG) Protects Non-Malignant Lung Cells from Radiation Damage

    doi: 10.3390/ijms17010007

    Figure Lengend Snippet: Evaluation of γ-Η2ΑΧ foci in irradiated murine epithelial cells, endothelial cells and wild-type fibroblasts. ( A ) Representative panels of immunofluorescence visualization of γ-H2AX foci (green) in murine lung epithelial cells at 200× magnification. Cells were pre-treated with SDG for 6 h and irradiated with γ-rays (2 Gy); ( B – D ) Evaluation of DNA damage by γ-Η2ΑΧ foci counting on ( B ) Epithelial Cells, ( C ) Endothelial Cells, and ( D ) Fibroblasts; ( E – G ) Flow cytometric (FACS) analysis of γ-H2AX foci in irradiated murine lung cells ( E ) Epithelial Cells, ( F ) Endothelial Cells, and ( G ) Fibroblasts. Cells were treated with SDG (50 μM) for 6 h and gamma-irradiated (2 Gy). At desired time interval, cells were processed for FACS analysis. Data were quantified using Summit software and is represented as mean ± SEM. * indicates p

    Article Snippet: P-Histone H2AX (rabbit mAb) was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

    Techniques: Irradiation, Immunofluorescence, Flow Cytometry, FACS, Software