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  • 97
    Millipore histone h1
    Histone H1, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1695 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam histone h3
    Plasma redox imbalance induces NETosis. a Concentration of free thiol in plasma from wild-type (WT) vs. albumin deficient (Alb −/− ) C57BL/6 mice and control vs. albumin-depleted plasma from NOD scid gamma (NSG) and BALB/c mice. b – d Plasma isolated from the blood of WT and Alb −/− C57BL/6 mice and NSG mice 2 days post saline, IAA (30 mg/kg), or LPS (8 mg/kg) injection was subjected to FPLC ( b , c ) and Western blotting ( d ). b Representative FPLC results for the redox state of albumin in C57BL/6 (left) and NSG (right) mice. c The ratio of peak intensity for reduced/oxidized albumin in NSG mice. d Western blotting with an antibody against C-reactive protein (CRP) and serum amyloid P component (SAP). e – g Plasma was isolated from the blood of WT and Alb −/− C57BL/6 mice and NSG mice 2 days post saline or IAA (30 mg/kg) injection following intravenous injection of PBS or murine albumin (20 mg/mouse). e , f Immunoprecipitation of cell-free DNA in the plasma from C57BL/6 ( e ) and NSG ( f ) mice using an anti-dsDNA antibody followed by Western blotting using an antibody against citrullinated <t>histone</t> H3 (Cit-H3). g Plasma free thiol concentration. a , c , g Results represent individual values with the mean ± s.d. ( n = 3–6; biological replicates, * P
    Histone H3, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 6925 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc histone h3
    JA2131 induces hyperPARylation of PARP1 . a  Subcellular PARG protein expression patterns in cultured cells. Subcellular fractionated lysates were immunoblotted with anti-PARG, (upper panel) followed by nuclear (N) marker anti- Laminin Subunit Beta 1 (LAMB1, upper middle panel), chromatin (Chr) marker anti-Histone H3 (H3, lower middle panel) and the cytoplasmic (C) marker anti- Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, lower panel)  b  PC3 cells were treated with DMSO or PARG inhibitor JA2131 (2131) for 2 h then irradiated with 7 Gy and allowed to recover for 0.5 h or 1.0 h before lysis and subcellular fractionation. Chromatin bound cell-extracts were analyzed with anti-PARP1 antibody (upper panel) followed by probing with total Histone H2AX (t-H2AX, lower panel) as the loading control.  c  DMSO or 2131-treated PC3 cells were irradiated and recovered for 2 h as above, chromatin fractions were then incubated with or without purified PARG enzyme (±PARG) for 30 min at 37 °C then immunoblotted with anti-PARP1 (upper panel) and then with anti-histone H3 (lower panel) as loading control.  d  Quantitative analysis of western blot of  b , where expression of PARP1 levels in DMSO treated was normalized to 1. High molecular weight (HMW) in red PARP1 is significantly decreased in the presence of purified truncated PARG protein. The low molecular weight in black PARP1 bands are not significantly affected,  n  = 3. Plus denotes added purified PARG enzyme. Source Data are provided as a Source Data file.
    Histone H3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 5201 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti histone h3 antibody nuclear loading control and chip grade
    JA2131 induces hyperPARylation of PARP1 . a  Subcellular PARG protein expression patterns in cultured cells. Subcellular fractionated lysates were immunoblotted with anti-PARG, (upper panel) followed by nuclear (N) marker anti- Laminin Subunit Beta 1 (LAMB1, upper middle panel), chromatin (Chr) marker anti-Histone H3 (H3, lower middle panel) and the cytoplasmic (C) marker anti- Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, lower panel)  b  PC3 cells were treated with DMSO or PARG inhibitor JA2131 (2131) for 2 h then irradiated with 7 Gy and allowed to recover for 0.5 h or 1.0 h before lysis and subcellular fractionation. Chromatin bound cell-extracts were analyzed with anti-PARP1 antibody (upper panel) followed by probing with total Histone H2AX (t-H2AX, lower panel) as the loading control.  c  DMSO or 2131-treated PC3 cells were irradiated and recovered for 2 h as above, chromatin fractions were then incubated with or without purified PARG enzyme (±PARG) for 30 min at 37 °C then immunoblotted with anti-PARP1 (upper panel) and then with anti-histone H3 (lower panel) as loading control.  d  Quantitative analysis of western blot of  b , where expression of PARP1 levels in DMSO treated was normalized to 1. High molecular weight (HMW) in red PARP1 is significantly decreased in the presence of purified truncated PARG protein. The low molecular weight in black PARP1 bands are not significantly affected,  n  = 3. Plus denotes added purified PARG enzyme. Source Data are provided as a Source Data file.
    Anti Histone H3 Antibody Nuclear Loading Control And Chip Grade, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 621 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Upstate Biotechnology Inc histone h2a
    Histone <t>H2A-AQE</t> mutant cells show defects in checkpoint signaling. (A) H2A-AQE mutations cause reduced Rad3-dependent phosphorylation of Chk1 and Crb2 in response to IR. Extracts were prepared from cells exposed to the indicated doses of IR immediately after irradiation and visualized by Western blotting. Arrows indicate phosphorylated forms of Chk1 and Crb2. Strains used were chk1 + (TMN2665), chk1-myc (TMN3309), chk1-myc hta1 hta2 (TMN3310), chk1-myc rad3 Δ (NR2648), crb2 Δ (LLD3339), crb2 + (KS1599), crb2 + hta1 hta2 (TMN3290), and crb2 + rad3 Δ (TMN2938). (B) H2A-AQE mutant cells prematurely reentered the cell cycle after irradiation with high-dose IR. Wild-type ( cdc25-22 ; TMN3311) or H2A-AQE mutant ( cdc25-22 hta1-S129A hta2-S128A ; TMN3312) cells were synchronized at 35°C for 2.5 h, exposed to the indicated doses of IR at room temperature, fixed with glutaraldehyde, and stained with calcoflour, and the percentage of septated cells was counted for each time point. A schematic drawing of the S. pombe cell cycle with the arrest points for cdc25-22 and adh-wee1-50 cells is indicated on the right. (C) Artificial cell cycle arrest of the H2-AQE mutant can rescue only a small fraction of cells from IR-induced DNA damage. The experimental scheme is drawn on the right. Cells expressing the constitutive temperature-sensitive allele ( wee1-50 ) of Cdc2 inhibitor Wee1 were arrested at 25°C 2 h prior to IR (1,200 Gy) and held arrested during irradiation. After completion of IR treatment, cells were plated at 25°C and then shifted to permissive temperature (36°C) at the indicated time. Wild-type ( adh-wee1-50 ; TMN2713) and hta1 hta2 adh-wee1-50 (TMN3421) cells were used.
    Histone H2a, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 91/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti histone h3
    BRE overexpression affects IR-induced CDC25A degradation. a DNA synthesis in ES cells after different doses of IR. b Western blot analysis of CDC25A and Tyr15 phosphorylation of CDK1 (p-CDK1) at different times after exposing the cells to 6 Gy IR in Brca2 CKO/KO ;MSCV-BRE cells compared to Brca2 CKO/KO cells. GAPDH was used as a loading control. Numbers below indicate the lane numbers. The quantification of CDC25A (middle panel) and p-CDK1 (lower panel) band intensity for each cell line is shown as histogram. Relative band intensities were calculated by dividing GAPDH-normalized CDC25A/p-CDK1 intensity at particular time point with GAPDH-normalized CDC25A/p-CDK1 intensity of corresponding untreated cells. c MCF7 cells with inducible HA-BRE expression cassette were transfected with CRE plasmid and subjected to 6 Gy IR after 48 h of transfection. Western blots of endogenous CDC25A in presence (lanes 4–6) and absence (lanes 1–3) of HA-BRE expression at different times after irradiation is shown in the upper panel. Lower panel shows the quantification of CDC25A band intensity normalized against GAPDH. d Immunoblot showing CDC25A degradation after IR treatment in MCF7 cells transfected either with non-specific (NS) siRNA or two different siRNAs against BRE (#1 and #2). <t>Histone</t> H3 was used as a loading control. Relative CDC25A band intensities were calculated by dividing GAPDH-normalized CDC25A intensity at a particular time point with GAPDH-normalized CDC25A intensity of untreated (0 h IR) cells of corresponding siRNA treatment. e Western blot showing IR-induced CDC25A degradation in MCF7 cells with stably integrated CRE inducible HA-BRE expression cassette after transfection either with non-specific (NS) siRNA or two different siRNAs against MERIT40 (#1 and #2) along with CRE -expressing plasmid. Non-specific (NS) siRNA transfection was used as control. Relative abundance of CDC25A at a particular time point was measured by comparing GAPDH-normalized band intensities at that point divided by GAPDH-normalized CDC25A intensity of untreated (0 h IR) cells of corresponding treatment. All histograms show the average of three independent experiments and error bars represent s.d. P values were calculated using paired two-tailed t
    Anti Histone H3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1887 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti histone h3 acetyl k27 antibody chip grade
    Enhancer profiling of maEpiSCs. ( A ) <t>H3K27ac</t> densities shown using a boxplot and ( B ) scatter plot (normalized tag density; log2 RPBM) at EpiSC-regions in conventional EpiSCs, maEpiSCs, MEFs, and ESCs. ( C ) H3K27ac densities shown using a boxplot at ESC-defined super-enhancers. ( D ) Analysis of H3K27ac levels in maEpiSCs, conventional EpiSCs, ESCs and MEFs using principal component analysis (PCA). ( E ) H3K27ac densities shown in heat maps at EpiSC-defined peaks. ( F ) Overlap between H3K27ac in maEpiSCs and ES cells (top left), maEpiSCs and conventional EpiSCs (middle left), and maEpiSCs and MEFs (bottom left) shown using Venn diagrams. ( G ) Venn diagrams of H3K4me2 levels between maEpiSCs and ES cells (top right), maEpiSCs and conventional EpiSCs (middle right), and maEpiSCs and MEFs (bottom right). ( H ) H3K4me2 densities shown using a boxplot and ( I ) scatter plots at EpiSC-defined regions. ( J ) H3K4me2 density shown using a boxplot at ESC-defined super-enhancers. ( K ) PCA of H3K4me2 levels between maEpiSCs, conventional EpiSCs, ES cells and MEFs. ( L ) Heat maps of H3K4me2 densities at transcriptional start site (TSS) regions. ( M ) Custom UCSC views of H3K4me2 and H3K27ac levels in conventional EpiSCs, maEpiSCs, MEFs, and ESCs.
    Anti Histone H3 Acetyl K27 Antibody Chip Grade, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 840 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti h3
    Characterization of micronuclei in t e × l s hybrid embryos and link to embryo death a, Disrupted micronuclei envelopes in t e × l s hybrid embryos. Whole mount embryo immunofluorescence was performed in t e × l s hybrid embryos using the YO-PRO DNA dye (top) and anti-Lamin B1 antibody (middle), corresponding channels are shown in green and magenta, respectively. The merged images are shown below. 25 micronuclei within 5 different embryos were analyzed. Intact (left) and disrupted (right) envelopes were observed in all analyzed embryos. Scale bar is 10 μm. b, DNA damage in t e × l s hybrid embryo micronuclei. Whole mount embryo immunofluorescence was performed in t e × l s hybrid embryos using <t>anti-histone</t> H3 (top) and anti-γH2A.X (middle) antibodies, corresponding channels are shown in green and magenta, respectively. The merge images are shown below. 21 micronuclei within different 6 embryos were analyzed. Micronuclei with undamaged (left; negative γH2A.X signal) and damaged (right; positive γH2A.X signal) DNA were observed in all analyzed embryos. Zoomed images of micronuclei are shown on the right of each image. Scale bar is 10 μm. c, TUNEL assay in apoptotic X. tropicalis and t e × l s hybrid embryos. X. tropicalis (left), X. tropicalis treated with cycloheximide (middle left) or hydroxyurea (middle right) as indicated, and t e × l s hybrid (right) embryos were prepared for TUNEL assay 5 hpf (equivalent stage 9; top), 7 hpf (equivalent stage 10; middle) and 9.5 hpf (equivalent stage 10.5; bottom). Identical results were obtained over 3 different experiments. Representative images are shown and were taken under identical conditions.
    Anti H3, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 3313 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam h3k4me3
    Characterization of micronuclei in t e × l s hybrid embryos and link to embryo death a, Disrupted micronuclei envelopes in t e × l s hybrid embryos. Whole mount embryo immunofluorescence was performed in t e × l s hybrid embryos using the YO-PRO DNA dye (top) and anti-Lamin B1 antibody (middle), corresponding channels are shown in green and magenta, respectively. The merged images are shown below. 25 micronuclei within 5 different embryos were analyzed. Intact (left) and disrupted (right) envelopes were observed in all analyzed embryos. Scale bar is 10 μm. b, DNA damage in t e × l s hybrid embryo micronuclei. Whole mount embryo immunofluorescence was performed in t e × l s hybrid embryos using <t>anti-histone</t> H3 (top) and anti-γH2A.X (middle) antibodies, corresponding channels are shown in green and magenta, respectively. The merge images are shown below. 21 micronuclei within different 6 embryos were analyzed. Micronuclei with undamaged (left; negative γH2A.X signal) and damaged (right; positive γH2A.X signal) DNA were observed in all analyzed embryos. Zoomed images of micronuclei are shown on the right of each image. Scale bar is 10 μm. c, TUNEL assay in apoptotic X. tropicalis and t e × l s hybrid embryos. X. tropicalis (left), X. tropicalis treated with cycloheximide (middle left) or hydroxyurea (middle right) as indicated, and t e × l s hybrid (right) embryos were prepared for TUNEL assay 5 hpf (equivalent stage 9; top), 7 hpf (equivalent stage 10; middle) and 9.5 hpf (equivalent stage 10.5; bottom). Identical results were obtained over 3 different experiments. Representative images are shown and were taken under identical conditions.
    H3k4me3, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 3837 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho histone h3
    RELB-P53 suppresses endothelial growth in the absence of PKM2. a EdU and <t>phospho-Histone</t> H3 positive cell numbers in control, RELB KD , PKM2 KD and PKM2 KD /RELB KD ECs ( n = 6). b Western blot analysis of RELB, P53, P21 and PKM2 expression in control, RELB KD , PKM2 KD and PKM2 KD /RELB KD ECs. c EdU and phospho-Histone H3 positive cell numbers in control, P53 KD , PKM2 KD and PKM2 KD /P53 KD ECs ( n = 12). d Western blot analysis of RELB, P53, P21 and PKM2 expression in control, P53 KD , PKM2 KD and PKM2 KD /P53 KD ECs. e mRNA expression levels of key enzymes for pyrimidine synthesis in control, P53 KD , PKM2 KD and PKM2 KD /P53 KD ECs (RNA-seq analysis, n = 3). f Relative N -carbamoyl aspartate levels, UTP levels, incorporation of [U-13C 6 ]-glucose into m+3 and m+5 UTP, and relative dTTP levels in control, P53 KD , PKM2 KD and PKM2 KD /P53 KD ECs ( n = 3). a , c Data represent means ± s.e.m., e , f data represent means ± s.d., m+ n represents the mass isotopomers for individual metabolites (*** P
    Phospho Histone H3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1255 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti acetyl histone h4
    miR-222 suppresses BRG1- and STAT-dependent inflammatory gene expression a effect on LPS-induced gene expression. b , ChIP in iBMDMs transduced with overexpression constructs (n=3 independent experiments; p-values from Students t-test for paired values, 2-sided). c-d , Schematic of treatments (c) and genes (d) analyzed in (e-i). e-f transcription factor motifs, and statistically over-represented gene ontology terms (determined by PANTHER) for indicated gene groups (n=103 gene expression values/group). g-h , Transcription factor occupancy (g) and <t>histone</t> modification (h) at promoters, quantified from published ChIP-seq datasets. i , ChIP for STAT2 occupancy in peritoneal macrophages. Values normalized to maximal binding detected for each ChIP (WT, miR-221/222 KO n=4 biologically independent samples; Stat1/2 KO n=2 biologically independent samples. p-values only calculated for WT vs. miR-221/222 KO comparisons by Students t-test, 2-sided, heteroscedastic). j , Model of miR-221/222 effect on chromatin at affected gene promoters. For all bar graphs and dot plots, center represents mean and error bars (if present) the SEM.
    Anti Acetyl Histone H4, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 890 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti histone
    miR-222 suppresses BRG1- and STAT-dependent inflammatory gene expression a effect on LPS-induced gene expression. b , ChIP in iBMDMs transduced with overexpression constructs (n=3 independent experiments; p-values from Students t-test for paired values, 2-sided). c-d , Schematic of treatments (c) and genes (d) analyzed in (e-i). e-f transcription factor motifs, and statistically over-represented gene ontology terms (determined by PANTHER) for indicated gene groups (n=103 gene expression values/group). g-h , Transcription factor occupancy (g) and <t>histone</t> modification (h) at promoters, quantified from published ChIP-seq datasets. i , ChIP for STAT2 occupancy in peritoneal macrophages. Values normalized to maximal binding detected for each ChIP (WT, miR-221/222 KO n=4 biologically independent samples; Stat1/2 KO n=2 biologically independent samples. p-values only calculated for WT vs. miR-221/222 KO comparisons by Students t-test, 2-sided, heteroscedastic). j , Model of miR-221/222 effect on chromatin at affected gene promoters. For all bar graphs and dot plots, center represents mean and error bars (if present) the SEM.
    Anti Histone, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 98 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti histone antibodies
    miR-222 suppresses BRG1- and STAT-dependent inflammatory gene expression a effect on LPS-induced gene expression. b , ChIP in iBMDMs transduced with overexpression constructs (n=3 independent experiments; p-values from Students t-test for paired values, 2-sided). c-d , Schematic of treatments (c) and genes (d) analyzed in (e-i). e-f transcription factor motifs, and statistically over-represented gene ontology terms (determined by PANTHER) for indicated gene groups (n=103 gene expression values/group). g-h , Transcription factor occupancy (g) and <t>histone</t> modification (h) at promoters, quantified from published ChIP-seq datasets. i , ChIP for STAT2 occupancy in peritoneal macrophages. Values normalized to maximal binding detected for each ChIP (WT, miR-221/222 KO n=4 biologically independent samples; Stat1/2 KO n=2 biologically independent samples. p-values only calculated for WT vs. miR-221/222 KO comparisons by Students t-test, 2-sided, heteroscedastic). j , Model of miR-221/222 effect on chromatin at affected gene promoters. For all bar graphs and dot plots, center represents mean and error bars (if present) the SEM.
    Anti Histone Antibodies, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 93 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam h3k9me3
    miR-222 suppresses BRG1- and STAT-dependent inflammatory gene expression a effect on LPS-induced gene expression. b , ChIP in iBMDMs transduced with overexpression constructs (n=3 independent experiments; p-values from Students t-test for paired values, 2-sided). c-d , Schematic of treatments (c) and genes (d) analyzed in (e-i). e-f transcription factor motifs, and statistically over-represented gene ontology terms (determined by PANTHER) for indicated gene groups (n=103 gene expression values/group). g-h , Transcription factor occupancy (g) and <t>histone</t> modification (h) at promoters, quantified from published ChIP-seq datasets. i , ChIP for STAT2 occupancy in peritoneal macrophages. Values normalized to maximal binding detected for each ChIP (WT, miR-221/222 KO n=4 biologically independent samples; Stat1/2 KO n=2 biologically independent samples. p-values only calculated for WT vs. miR-221/222 KO comparisons by Students t-test, 2-sided, heteroscedastic). j , Model of miR-221/222 effect on chromatin at affected gene promoters. For all bar graphs and dot plots, center represents mean and error bars (if present) the SEM.
    H3k9me3, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 3051 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore phospho histone h3
    Piezo + /EP cells are ISC-derived EE precursors with reduced mitotic ability. a. Midguts from flies treated with Bleomycin (10 μg/ml Bleo in 5% sucrose) or the γ-secretase inhibitor DAPT (4 mM DAPT in 5% sucrose). Cells that are positive for both Piezo and Pros are indicated by arrowheads. Note that the majority ( > 95%) of Piezo and Pros double positive cells are also positive for esg, suggesting that these cells are “newborn” EEs that still retain esg-GFP signal. b. Percentage of the newborn EEs (Piezo and Pros double positive cells vs. total Pros + EEs) in fly midguts under control, Bleomycin, and DAPT treatments. Cells within 200 μm X 200 μm areas, n=27 (control), n=25 (Bleo), and n=22 (DAPT), were analyzed. c. Midgut with stem cells labeled by esg-GFP (Green), Piezo + cells labeled by Piezo-Gal4 > nlsRFP (Red), and mitotic cells labeled by <t>anti-phospho-Histone</t> H3 (pH3) (Magenta). pH3 + mitotic cell is indicated by arrowhead. d,e. Representative images of midguts from flies feed on either control (5% sucrose) or Bleomycin (5% sucrose plus 10ug/ml Bleomycin) food. Piezo + EP cells are labeled by Piezo-Gal4 > nlsGFP (Green), mitotic cells are labeled by pH3 staining (Red). Mitotic Piezo + cells are indicated by arrowheads. Since all pH3 + cells are Dl + cells (according to Dl-lacZ labeled midgut), we counted all Piezo negative pH3 + cells as pH3 + ISCs. Under both control (5% sucrose) and damage (5% sucrose + 10 μg/ml Bleomycin) conditions, only around 8–10% of the pH3 + cells are Piezo + (~40% of total Dl + cells), suggesting that Piezo + cells are significantly less mitotically active compared to Piezo- Dl + cells. f. If the pH3 + Piezo + cells are also Pros + . Around 50% of these pH3 + Piezo + cells show low levels of Pros staining. Meanwhile, all the pH3 + Pros + cells are positive for Piezo, suggesting that Piezo + EP cells represent more general EE precursor cells compared to EMCs. Mitotic Piezo + cells are indicated by arrowheads. All experiments were independently repeated at least twice with similar results presented in the figures. g. Random GFP + clones were generated using hsFlp; Ubi-(FRT.Stop)GFP/Piezo-Gal4; UAS-nlsRFP . 3–4 days old flies were heat shocked at 37°C for 30 min once to induce clones in ISCs. Then these flies were kept at 25°C for 2 weeks before analysis. Within each GFP + clone, which is derived from ISCs, there are typically 1–2 Piezo + cells in the cluster (indicated by arrowheads), suggesting that Piezo + cells are generated from ISCs after adulthood. All experiments were independently repeated at least twice with similar results presented in the figures. Data are expressed as mean + s.e.m. P-values are calculated from two-tailed Student t-test with unequal variance. Scale bar: a,c , 50 μm; d,f 20 μm; g , 25 μm.
    Phospho Histone H3, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1188 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam h3k4me1
    Piezo + /EP cells are ISC-derived EE precursors with reduced mitotic ability. a. Midguts from flies treated with Bleomycin (10 μg/ml Bleo in 5% sucrose) or the γ-secretase inhibitor DAPT (4 mM DAPT in 5% sucrose). Cells that are positive for both Piezo and Pros are indicated by arrowheads. Note that the majority ( > 95%) of Piezo and Pros double positive cells are also positive for esg, suggesting that these cells are “newborn” EEs that still retain esg-GFP signal. b. Percentage of the newborn EEs (Piezo and Pros double positive cells vs. total Pros + EEs) in fly midguts under control, Bleomycin, and DAPT treatments. Cells within 200 μm X 200 μm areas, n=27 (control), n=25 (Bleo), and n=22 (DAPT), were analyzed. c. Midgut with stem cells labeled by esg-GFP (Green), Piezo + cells labeled by Piezo-Gal4 > nlsRFP (Red), and mitotic cells labeled by <t>anti-phospho-Histone</t> H3 (pH3) (Magenta). pH3 + mitotic cell is indicated by arrowhead. d,e. Representative images of midguts from flies feed on either control (5% sucrose) or Bleomycin (5% sucrose plus 10ug/ml Bleomycin) food. Piezo + EP cells are labeled by Piezo-Gal4 > nlsGFP (Green), mitotic cells are labeled by pH3 staining (Red). Mitotic Piezo + cells are indicated by arrowheads. Since all pH3 + cells are Dl + cells (according to Dl-lacZ labeled midgut), we counted all Piezo negative pH3 + cells as pH3 + ISCs. Under both control (5% sucrose) and damage (5% sucrose + 10 μg/ml Bleomycin) conditions, only around 8–10% of the pH3 + cells are Piezo + (~40% of total Dl + cells), suggesting that Piezo + cells are significantly less mitotically active compared to Piezo- Dl + cells. f. If the pH3 + Piezo + cells are also Pros + . Around 50% of these pH3 + Piezo + cells show low levels of Pros staining. Meanwhile, all the pH3 + Pros + cells are positive for Piezo, suggesting that Piezo + EP cells represent more general EE precursor cells compared to EMCs. Mitotic Piezo + cells are indicated by arrowheads. All experiments were independently repeated at least twice with similar results presented in the figures. g. Random GFP + clones were generated using hsFlp; Ubi-(FRT.Stop)GFP/Piezo-Gal4; UAS-nlsRFP . 3–4 days old flies were heat shocked at 37°C for 30 min once to induce clones in ISCs. Then these flies were kept at 25°C for 2 weeks before analysis. Within each GFP + clone, which is derived from ISCs, there are typically 1–2 Piezo + cells in the cluster (indicated by arrowheads), suggesting that Piezo + cells are generated from ISCs after adulthood. All experiments were independently repeated at least twice with similar results presented in the figures. Data are expressed as mean + s.e.m. P-values are calculated from two-tailed Student t-test with unequal variance. Scale bar: a,c , 50 μm; d,f 20 μm; g , 25 μm.
    H3k4me1, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 1918 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    h3k4me1 - by Bioz Stars, 2021-01
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    96
    Millipore hdac3
    <t>Hdac3</t> overexpression inhibits cyclin-dependent kinase (CDK) inhibitors Cdkn1a (p21 WAF / CIP1 ), Cdkn1b (p27 Kip1 ), Cdkn1c (p57 Kip2 ), Cdkn2c (p18 INC4c ), Cdkn2b (p15 INC4b ), and Cdkn2a ( p16 INC4a ) . A and B , transcript for Cdkn1a , Cdkn1b , Cdkn1c , Cdkn2c , Cdkn2b, and Cdkn2a were detected by qRT-PCR in hearts from P1 ( A ) and 2-month-old ( B ) wild-type and Hdac3 -Tg mice. Three mice in each group were tested, and values are expressed as the -fold change in transcript abundance (±S.D.) when compared with wild-type mice. N.S. , not significant.
    Hdac3, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 392 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    A nucleosome is the basic repeating unit of chromatin in which 146 base pairs of DNA wrap twice around a histone octamer consisting of two copies of each of the
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    The nucleosome is made up of four core histone proteins H2A H2B H3 and H4 and is the primary building block of chromatin The N terminal tail of core histones
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    N/A
    A nucleosome is the basic repeating unit of chromatin in which 146 base pairs of DNA wrap twice around a histone octamer consisting of two copies of each of the
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    N/A
    A nucleosome is the basic repeating unit of chromatin in which 146 base pairs of DNA wrap twice around a histone octamer consisting of two copies of each of the
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    Image Search Results


    Plasma redox imbalance induces NETosis. a Concentration of free thiol in plasma from wild-type (WT) vs. albumin deficient (Alb −/− ) C57BL/6 mice and control vs. albumin-depleted plasma from NOD scid gamma (NSG) and BALB/c mice. b – d Plasma isolated from the blood of WT and Alb −/− C57BL/6 mice and NSG mice 2 days post saline, IAA (30 mg/kg), or LPS (8 mg/kg) injection was subjected to FPLC ( b , c ) and Western blotting ( d ). b Representative FPLC results for the redox state of albumin in C57BL/6 (left) and NSG (right) mice. c The ratio of peak intensity for reduced/oxidized albumin in NSG mice. d Western blotting with an antibody against C-reactive protein (CRP) and serum amyloid P component (SAP). e – g Plasma was isolated from the blood of WT and Alb −/− C57BL/6 mice and NSG mice 2 days post saline or IAA (30 mg/kg) injection following intravenous injection of PBS or murine albumin (20 mg/mouse). e , f Immunoprecipitation of cell-free DNA in the plasma from C57BL/6 ( e ) and NSG ( f ) mice using an anti-dsDNA antibody followed by Western blotting using an antibody against citrullinated histone H3 (Cit-H3). g Plasma free thiol concentration. a , c , g Results represent individual values with the mean ± s.d. ( n = 3–6; biological replicates, * P

    Journal: Nature Communications

    Article Title: Plasma redox imbalance caused by albumin oxidation promotes lung-predominant NETosis and pulmonary cancer metastasis

    doi: 10.1038/s41467-018-07550-x

    Figure Lengend Snippet: Plasma redox imbalance induces NETosis. a Concentration of free thiol in plasma from wild-type (WT) vs. albumin deficient (Alb −/− ) C57BL/6 mice and control vs. albumin-depleted plasma from NOD scid gamma (NSG) and BALB/c mice. b – d Plasma isolated from the blood of WT and Alb −/− C57BL/6 mice and NSG mice 2 days post saline, IAA (30 mg/kg), or LPS (8 mg/kg) injection was subjected to FPLC ( b , c ) and Western blotting ( d ). b Representative FPLC results for the redox state of albumin in C57BL/6 (left) and NSG (right) mice. c The ratio of peak intensity for reduced/oxidized albumin in NSG mice. d Western blotting with an antibody against C-reactive protein (CRP) and serum amyloid P component (SAP). e – g Plasma was isolated from the blood of WT and Alb −/− C57BL/6 mice and NSG mice 2 days post saline or IAA (30 mg/kg) injection following intravenous injection of PBS or murine albumin (20 mg/mouse). e , f Immunoprecipitation of cell-free DNA in the plasma from C57BL/6 ( e ) and NSG ( f ) mice using an anti-dsDNA antibody followed by Western blotting using an antibody against citrullinated histone H3 (Cit-H3). g Plasma free thiol concentration. a , c , g Results represent individual values with the mean ± s.d. ( n = 3–6; biological replicates, * P

    Article Snippet: We used LL-37 (1:1,000, OSC00009W, Osenses Pty Ltd., Australia), histone H3 (citrulline R2 + R8 + R17, 1:500, ab5103, Abcam, MA, USA), transferrin (1:500, ab82411, Abcam, MA, USA), β-actin (1:200, sc-47778 HRP, Santa Cruz, CA, USA) where indicated.

    Techniques: Concentration Assay, Mouse Assay, Isolation, Injection, Fast Protein Liquid Chromatography, Western Blot, Immunoprecipitation

    Chloroquine inhibits NET formation in acute pancreatitis. Neutrophils harvested from mice with pancreatitis had evidence of spontaneous NET formation and greater propensity to form NETs upon stimulation with platelet activating factor (PAF), as visualized by staining of DNA with Hoechst (A) . CQ treatment dramatically reduced spontaneous and stimulated NET formation. Supernatant DNA was measured to objectively quantify NETs (B) . Serum cell-free DNA (C) and citrullinated histone H3 (CitH3) (D) , biomarkers of in vivo NET formation were elevated with induction of pancreatitis, but significantly reduced with chloroquine treatment. Co-localization of pancreatic CitH3 (Green), with neutrophils (Ly-6G, Red) is increased in pancreatitis mice compared to sham controls (E) , but treatment with CQ reduced neutrophil CitH3 expression (20x, scale bar = 50 μm). These are representative images from at least two independent analyses. * p

    Journal: Frontiers in Immunology

    Article Title: Enhanced Neutrophil Extracellular Trap Formation in Acute Pancreatitis Contributes to Disease Severity and Is Reduced by Chloroquine

    doi: 10.3389/fimmu.2019.00028

    Figure Lengend Snippet: Chloroquine inhibits NET formation in acute pancreatitis. Neutrophils harvested from mice with pancreatitis had evidence of spontaneous NET formation and greater propensity to form NETs upon stimulation with platelet activating factor (PAF), as visualized by staining of DNA with Hoechst (A) . CQ treatment dramatically reduced spontaneous and stimulated NET formation. Supernatant DNA was measured to objectively quantify NETs (B) . Serum cell-free DNA (C) and citrullinated histone H3 (CitH3) (D) , biomarkers of in vivo NET formation were elevated with induction of pancreatitis, but significantly reduced with chloroquine treatment. Co-localization of pancreatic CitH3 (Green), with neutrophils (Ly-6G, Red) is increased in pancreatitis mice compared to sham controls (E) , but treatment with CQ reduced neutrophil CitH3 expression (20x, scale bar = 50 μm). These are representative images from at least two independent analyses. * p

    Article Snippet: The sections were incubated with anti-Histone H3 (Rabbit, 1:200, citrulline R2 + R8 + R17, Abcam, ab5103) and anti-Ly6G (Rat, 1:100, RB6-8C5, Invitrogen).

    Techniques: Mouse Assay, Staining, In Vivo, Expressing

    JA2131 induces hyperPARylation of PARP1 . a  Subcellular PARG protein expression patterns in cultured cells. Subcellular fractionated lysates were immunoblotted with anti-PARG, (upper panel) followed by nuclear (N) marker anti- Laminin Subunit Beta 1 (LAMB1, upper middle panel), chromatin (Chr) marker anti-Histone H3 (H3, lower middle panel) and the cytoplasmic (C) marker anti- Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, lower panel)  b  PC3 cells were treated with DMSO or PARG inhibitor JA2131 (2131) for 2 h then irradiated with 7 Gy and allowed to recover for 0.5 h or 1.0 h before lysis and subcellular fractionation. Chromatin bound cell-extracts were analyzed with anti-PARP1 antibody (upper panel) followed by probing with total Histone H2AX (t-H2AX, lower panel) as the loading control.  c  DMSO or 2131-treated PC3 cells were irradiated and recovered for 2 h as above, chromatin fractions were then incubated with or without purified PARG enzyme (±PARG) for 30 min at 37 °C then immunoblotted with anti-PARP1 (upper panel) and then with anti-histone H3 (lower panel) as loading control.  d  Quantitative analysis of western blot of  b , where expression of PARP1 levels in DMSO treated was normalized to 1. High molecular weight (HMW) in red PARP1 is significantly decreased in the presence of purified truncated PARG protein. The low molecular weight in black PARP1 bands are not significantly affected,  n  = 3. Plus denotes added purified PARG enzyme. Source Data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Selective small molecule PARG inhibitor causes replication fork stalling and cancer cell death

    doi: 10.1038/s41467-019-13508-4

    Figure Lengend Snippet: JA2131 induces hyperPARylation of PARP1 . a Subcellular PARG protein expression patterns in cultured cells. Subcellular fractionated lysates were immunoblotted with anti-PARG, (upper panel) followed by nuclear (N) marker anti- Laminin Subunit Beta 1 (LAMB1, upper middle panel), chromatin (Chr) marker anti-Histone H3 (H3, lower middle panel) and the cytoplasmic (C) marker anti- Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, lower panel) b PC3 cells were treated with DMSO or PARG inhibitor JA2131 (2131) for 2 h then irradiated with 7 Gy and allowed to recover for 0.5 h or 1.0 h before lysis and subcellular fractionation. Chromatin bound cell-extracts were analyzed with anti-PARP1 antibody (upper panel) followed by probing with total Histone H2AX (t-H2AX, lower panel) as the loading control. c DMSO or 2131-treated PC3 cells were irradiated and recovered for 2 h as above, chromatin fractions were then incubated with or without purified PARG enzyme (±PARG) for 30 min at 37 °C then immunoblotted with anti-PARP1 (upper panel) and then with anti-histone H3 (lower panel) as loading control. d Quantitative analysis of western blot of b , where expression of PARP1 levels in DMSO treated was normalized to 1. High molecular weight (HMW) in red PARP1 is significantly decreased in the presence of purified truncated PARG protein. The low molecular weight in black PARP1 bands are not significantly affected, n  = 3. Plus denotes added purified PARG enzyme. Source Data are provided as a Source Data file.

    Article Snippet: Primary antibodies were purchased from the following sources; Actin, GAPDH, γH2AX and Histone-H3 was from Cell Signaling Technology.

    Techniques: Expressing, Cell Culture, Marker, Irradiation, Lysis, Fractionation, Incubation, Purification, Western Blot, Molecular Weight

    Histone H2A-AQE mutant cells show defects in checkpoint signaling. (A) H2A-AQE mutations cause reduced Rad3-dependent phosphorylation of Chk1 and Crb2 in response to IR. Extracts were prepared from cells exposed to the indicated doses of IR immediately after irradiation and visualized by Western blotting. Arrows indicate phosphorylated forms of Chk1 and Crb2. Strains used were chk1 + (TMN2665), chk1-myc (TMN3309), chk1-myc hta1 hta2 (TMN3310), chk1-myc rad3 Δ (NR2648), crb2 Δ (LLD3339), crb2 + (KS1599), crb2 + hta1 hta2 (TMN3290), and crb2 + rad3 Δ (TMN2938). (B) H2A-AQE mutant cells prematurely reentered the cell cycle after irradiation with high-dose IR. Wild-type ( cdc25-22 ; TMN3311) or H2A-AQE mutant ( cdc25-22 hta1-S129A hta2-S128A ; TMN3312) cells were synchronized at 35°C for 2.5 h, exposed to the indicated doses of IR at room temperature, fixed with glutaraldehyde, and stained with calcoflour, and the percentage of septated cells was counted for each time point. A schematic drawing of the S. pombe cell cycle with the arrest points for cdc25-22 and adh-wee1-50 cells is indicated on the right. (C) Artificial cell cycle arrest of the H2-AQE mutant can rescue only a small fraction of cells from IR-induced DNA damage. The experimental scheme is drawn on the right. Cells expressing the constitutive temperature-sensitive allele ( wee1-50 ) of Cdc2 inhibitor Wee1 were arrested at 25°C 2 h prior to IR (1,200 Gy) and held arrested during irradiation. After completion of IR treatment, cells were plated at 25°C and then shifted to permissive temperature (36°C) at the indicated time. Wild-type ( adh-wee1-50 ; TMN2713) and hta1 hta2 adh-wee1-50 (TMN3421) cells were used.

    Journal: Molecular and Cellular Biology

    Article Title: Histone H2A Phosphorylation Controls Crb2 Recruitment at DNA Breaks, Maintains Checkpoint Arrest, and Influences DNA Repair in Fission Yeast †

    doi: 10.1128/MCB.24.14.6215-6230.2004

    Figure Lengend Snippet: Histone H2A-AQE mutant cells show defects in checkpoint signaling. (A) H2A-AQE mutations cause reduced Rad3-dependent phosphorylation of Chk1 and Crb2 in response to IR. Extracts were prepared from cells exposed to the indicated doses of IR immediately after irradiation and visualized by Western blotting. Arrows indicate phosphorylated forms of Chk1 and Crb2. Strains used were chk1 + (TMN2665), chk1-myc (TMN3309), chk1-myc hta1 hta2 (TMN3310), chk1-myc rad3 Δ (NR2648), crb2 Δ (LLD3339), crb2 + (KS1599), crb2 + hta1 hta2 (TMN3290), and crb2 + rad3 Δ (TMN2938). (B) H2A-AQE mutant cells prematurely reentered the cell cycle after irradiation with high-dose IR. Wild-type ( cdc25-22 ; TMN3311) or H2A-AQE mutant ( cdc25-22 hta1-S129A hta2-S128A ; TMN3312) cells were synchronized at 35°C for 2.5 h, exposed to the indicated doses of IR at room temperature, fixed with glutaraldehyde, and stained with calcoflour, and the percentage of septated cells was counted for each time point. A schematic drawing of the S. pombe cell cycle with the arrest points for cdc25-22 and adh-wee1-50 cells is indicated on the right. (C) Artificial cell cycle arrest of the H2-AQE mutant can rescue only a small fraction of cells from IR-induced DNA damage. The experimental scheme is drawn on the right. Cells expressing the constitutive temperature-sensitive allele ( wee1-50 ) of Cdc2 inhibitor Wee1 were arrested at 25°C 2 h prior to IR (1,200 Gy) and held arrested during irradiation. After completion of IR treatment, cells were plated at 25°C and then shifted to permissive temperature (36°C) at the indicated time. Wild-type ( adh-wee1-50 ; TMN2713) and hta1 hta2 adh-wee1-50 (TMN3421) cells were used.

    Article Snippet: For visualization of phosphorylated histone H2A by indirect immunofluorescence microscopy, cells were fixed with 3.7% formaldehyde and processed as previously described ( ) using anti-phospho-H2AX (serine-139) polyclonal immunoglobulin G (IgG; Upstate Biotechnology) and Alexa Fluor 488 goat anti-rabbit IgG (Molecular Probes) as primary and secondary antibodies, respectively.

    Techniques: Mutagenesis, Irradiation, Western Blot, Staining, Expressing

    γ-H2A mediates accumulation of Crb2 at sites of DNA DSBs. (A) YFP-Crb2 focus formation is dependent on γ-H2A generated by Rad3 and Tel1 kinases. YFP-Crb2 localization was observed immediately after irradiation with IR (36 Gy). Strains used were wild type (LLD3260), hta1 hta2 (LLD3340), rad3 Δ tel1 Δ (LLD3341), rad3 Δ (LLD3342), and tel1 Δ (LLD3343). (B) Time-dependent change in Crb2 foci after exposure to IR (36 Gy). The number of cells with Crb2 foci at various times after IR treatment was counted and plotted as a percentage of cells with foci. (C) In vitro binding of partially purified Crb2 protein (from LLD3344) to the phosphorylated H2A C-tail peptide. Phosphorylated or unphosphorylated H2A C-tail peptide was conjugated to agarose beads and incubated with Crb2 protein prepared by two-step purification with IgG and calmodulin beads. Peptide-bound Crb2 was then visualized by Western blotting using polyclonal anti-Crb2 antibody. (D) IRIF formation by Rad26 and Rad32 is not affected by H2A-AQE mutations. Cells were irradiated with 180 Gy for YFP-Rad26 cells (TMN3337 and TMN3338) and 120 Gy for Rad32-GFP cells (LLD3335 and TMN3336) and observed immediately after irradiation.

    Journal: Molecular and Cellular Biology

    Article Title: Histone H2A Phosphorylation Controls Crb2 Recruitment at DNA Breaks, Maintains Checkpoint Arrest, and Influences DNA Repair in Fission Yeast †

    doi: 10.1128/MCB.24.14.6215-6230.2004

    Figure Lengend Snippet: γ-H2A mediates accumulation of Crb2 at sites of DNA DSBs. (A) YFP-Crb2 focus formation is dependent on γ-H2A generated by Rad3 and Tel1 kinases. YFP-Crb2 localization was observed immediately after irradiation with IR (36 Gy). Strains used were wild type (LLD3260), hta1 hta2 (LLD3340), rad3 Δ tel1 Δ (LLD3341), rad3 Δ (LLD3342), and tel1 Δ (LLD3343). (B) Time-dependent change in Crb2 foci after exposure to IR (36 Gy). The number of cells with Crb2 foci at various times after IR treatment was counted and plotted as a percentage of cells with foci. (C) In vitro binding of partially purified Crb2 protein (from LLD3344) to the phosphorylated H2A C-tail peptide. Phosphorylated or unphosphorylated H2A C-tail peptide was conjugated to agarose beads and incubated with Crb2 protein prepared by two-step purification with IgG and calmodulin beads. Peptide-bound Crb2 was then visualized by Western blotting using polyclonal anti-Crb2 antibody. (D) IRIF formation by Rad26 and Rad32 is not affected by H2A-AQE mutations. Cells were irradiated with 180 Gy for YFP-Rad26 cells (TMN3337 and TMN3338) and 120 Gy for Rad32-GFP cells (LLD3335 and TMN3336) and observed immediately after irradiation.

    Article Snippet: For visualization of phosphorylated histone H2A by indirect immunofluorescence microscopy, cells were fixed with 3.7% formaldehyde and processed as previously described ( ) using anti-phospho-H2AX (serine-139) polyclonal immunoglobulin G (IgG; Upstate Biotechnology) and Alexa Fluor 488 goat anti-rabbit IgG (Molecular Probes) as primary and secondary antibodies, respectively.

    Techniques: Generated, Irradiation, In Vitro, Binding Assay, Purification, Incubation, Western Blot

    Histone H2A C-tail SQE motif is important for survival in response to DNA damage. (A) Fivefold serial dilutions of wild-type and various histone H2A-AQE mutant S. pombe cells ( hta1-S129A and/or hta2-S128A ) were plated onto YES medium with the indicated concentrations of HU, MMS, or CPT. Pictures were taken after 4 days at 32°C. Strains used were hta1-S129A (TMN3294), hta2-S128A (TMN3295), hta1-S129A hta2-S128A (TMN3292 and TMN3293), and wild type (TMN2665). (B) Survival of wild-type and H2A-AQE mutant cells after exposure to the indicated doses of IR, UV, or incubation with 5 mU of bleomycin/ml for the indicated lengths of time. Strains used were wild type (TMN3296) and hta1-S129A hta2-S128A (TMN3293). Error bars indicate standard deviations from at least three experiments.

    Journal: Molecular and Cellular Biology

    Article Title: Histone H2A Phosphorylation Controls Crb2 Recruitment at DNA Breaks, Maintains Checkpoint Arrest, and Influences DNA Repair in Fission Yeast †

    doi: 10.1128/MCB.24.14.6215-6230.2004

    Figure Lengend Snippet: Histone H2A C-tail SQE motif is important for survival in response to DNA damage. (A) Fivefold serial dilutions of wild-type and various histone H2A-AQE mutant S. pombe cells ( hta1-S129A and/or hta2-S128A ) were plated onto YES medium with the indicated concentrations of HU, MMS, or CPT. Pictures were taken after 4 days at 32°C. Strains used were hta1-S129A (TMN3294), hta2-S128A (TMN3295), hta1-S129A hta2-S128A (TMN3292 and TMN3293), and wild type (TMN2665). (B) Survival of wild-type and H2A-AQE mutant cells after exposure to the indicated doses of IR, UV, or incubation with 5 mU of bleomycin/ml for the indicated lengths of time. Strains used were wild type (TMN3296) and hta1-S129A hta2-S128A (TMN3293). Error bars indicate standard deviations from at least three experiments.

    Article Snippet: For visualization of phosphorylated histone H2A by indirect immunofluorescence microscopy, cells were fixed with 3.7% formaldehyde and processed as previously described ( ) using anti-phospho-H2AX (serine-139) polyclonal immunoglobulin G (IgG; Upstate Biotechnology) and Alexa Fluor 488 goat anti-rabbit IgG (Molecular Probes) as primary and secondary antibodies, respectively.

    Techniques: Mutagenesis, Cycling Probe Technology, Incubation

    H2A-AQE cells show defects in HU-induced cell cycle arrest in the absence of S-phase checkpoint proteins. (A) H2A-AQE mutations make cells with mutations in the S-phase checkpoint ( mrc1 Δ and cds1 Δ), but not those with mutations in the DNA damage checkpoint ( crb2 Δ and chk1 Δ), more sensitive to HU. Viability of cells with indicated genotypes was measured by counting colony formation on YES plates after treatment with 12 mM HU for the indicated lengths of time. Strains used were wild type (TMN2665), hta1 hta2 (TMN3293), crb2 Δ (TMN2941), crb2 Δ hta1 hta2 (TMN3297), chk1 Δ (TMN2943), chk1 Δ hta1 hta2 (TMN3298), mrc1 Δ (TMN3326), mrc1 Δ hta1 hta2 (TMN3327), cds1 Δ (TMN2945), cds1 Δ hta1 hta2 (TMN3324), mrc1 Δ chk1 Δ (TMN3328), and cds1 Δ chk1 Δ (TMN3325). (B) Cells of the indicated genotypes (the same strains as in panel A) were mock treated or treated with 12 mM HU for 6 h and then stained with DAPI. Increased occurrence of the cut phenotype in mrc1 Δ hta1-S129A hta2-S128A and cds1 Δ hta1-S129A hta2-S128A cells, but not in chk1 Δ hta1-S129A hta2-S128A cells, is consistent with a defect in DNA damage checkpoint enforced by Chk1. (C) Fivefold serial dilutions of mrc1 Δ and mrc1 Δ hta1-S129A hta2-S128A cells carrying either the control pAL-SK+ plasmid or the Cds1 overexpression plasmid pAL-Cds1 (TMN3329, TMN3330, TMN3331, and TMN3332) were plated on minimal medium with the indicated concentrations of HU. Pictures were taken after 6 days at 32°C. (D) Fivefold serial dilutions of cells with the indicated genotypes (the same strains as used in panel A) were plated onto YES with the indicated concentrations of CPT. Pictures were taken after 4 days at 32°C.

    Journal: Molecular and Cellular Biology

    Article Title: Histone H2A Phosphorylation Controls Crb2 Recruitment at DNA Breaks, Maintains Checkpoint Arrest, and Influences DNA Repair in Fission Yeast †

    doi: 10.1128/MCB.24.14.6215-6230.2004

    Figure Lengend Snippet: H2A-AQE cells show defects in HU-induced cell cycle arrest in the absence of S-phase checkpoint proteins. (A) H2A-AQE mutations make cells with mutations in the S-phase checkpoint ( mrc1 Δ and cds1 Δ), but not those with mutations in the DNA damage checkpoint ( crb2 Δ and chk1 Δ), more sensitive to HU. Viability of cells with indicated genotypes was measured by counting colony formation on YES plates after treatment with 12 mM HU for the indicated lengths of time. Strains used were wild type (TMN2665), hta1 hta2 (TMN3293), crb2 Δ (TMN2941), crb2 Δ hta1 hta2 (TMN3297), chk1 Δ (TMN2943), chk1 Δ hta1 hta2 (TMN3298), mrc1 Δ (TMN3326), mrc1 Δ hta1 hta2 (TMN3327), cds1 Δ (TMN2945), cds1 Δ hta1 hta2 (TMN3324), mrc1 Δ chk1 Δ (TMN3328), and cds1 Δ chk1 Δ (TMN3325). (B) Cells of the indicated genotypes (the same strains as in panel A) were mock treated or treated with 12 mM HU for 6 h and then stained with DAPI. Increased occurrence of the cut phenotype in mrc1 Δ hta1-S129A hta2-S128A and cds1 Δ hta1-S129A hta2-S128A cells, but not in chk1 Δ hta1-S129A hta2-S128A cells, is consistent with a defect in DNA damage checkpoint enforced by Chk1. (C) Fivefold serial dilutions of mrc1 Δ and mrc1 Δ hta1-S129A hta2-S128A cells carrying either the control pAL-SK+ plasmid or the Cds1 overexpression plasmid pAL-Cds1 (TMN3329, TMN3330, TMN3331, and TMN3332) were plated on minimal medium with the indicated concentrations of HU. Pictures were taken after 6 days at 32°C. (D) Fivefold serial dilutions of cells with the indicated genotypes (the same strains as used in panel A) were plated onto YES with the indicated concentrations of CPT. Pictures were taken after 4 days at 32°C.

    Article Snippet: For visualization of phosphorylated histone H2A by indirect immunofluorescence microscopy, cells were fixed with 3.7% formaldehyde and processed as previously described ( ) using anti-phospho-H2AX (serine-139) polyclonal immunoglobulin G (IgG; Upstate Biotechnology) and Alexa Fluor 488 goat anti-rabbit IgG (Molecular Probes) as primary and secondary antibodies, respectively.

    Techniques: Staining, Plasmid Preparation, Over Expression, Cycling Probe Technology

    Genetic interactions of histone H2A-AQE mutations with mutations in DNA damage checkpoint genes. (A to D) Survival of cells carrying various combinations of mutations in H2A ( hta1-S129A and hta2-S128A , indicated as hta1 hta2 ), checkpoint ( rad3 Δ, crb2 Δ, and chk1 Δ), and DNA repair ( rqh1 Δ) genes after exposure to the indicated dose of IR. Strains used were rad3 Δ (TMN2947), rad3 Δ hta1 hta2 (TMN3299), rad3 Δ crb2 Δ (TMN3300), crb2 Δ (TMN2941), crb2 Δ hta1 hta2 (TMN3297), chk1 Δ (TMN2943), chk1 Δ hta1 hta2 (TMN3298), crb2 Δ chk1 Δ (TMN3305), crb2 Δ chk1 Δ hta1 hta2 (TMN3308), rqh1 Δ (TMN3301), rqh1 Δ hta1 hta2 (TMN3302), crb2 Δ rqh1 Δ (TMN3303), crb2 Δ rqh1 Δ hta1 hta2 (TMN3307), chk1 Δ rqh1 Δ (TMN3304), chk1 Δ rqh1 Δ hta1 hta2 (TMN3422), and crb2 Δ chk1 Δ rqh1 Δ (TMN3306). Error bars indicate standard deviations from at least three experiments. (E) Model depicting the γ-H2A-dependent modulation of Crb2 functions involving Chk1 and Rqh1, based on genetic interactions of IR sensitivity. (F) Model for the mammalian γ-H2AX-dependent DNA damage response. A speculative negative role of the γ-H2AX in DNA repair is indicated as a gray inhibitory sign covered with question marks.

    Journal: Molecular and Cellular Biology

    Article Title: Histone H2A Phosphorylation Controls Crb2 Recruitment at DNA Breaks, Maintains Checkpoint Arrest, and Influences DNA Repair in Fission Yeast †

    doi: 10.1128/MCB.24.14.6215-6230.2004

    Figure Lengend Snippet: Genetic interactions of histone H2A-AQE mutations with mutations in DNA damage checkpoint genes. (A to D) Survival of cells carrying various combinations of mutations in H2A ( hta1-S129A and hta2-S128A , indicated as hta1 hta2 ), checkpoint ( rad3 Δ, crb2 Δ, and chk1 Δ), and DNA repair ( rqh1 Δ) genes after exposure to the indicated dose of IR. Strains used were rad3 Δ (TMN2947), rad3 Δ hta1 hta2 (TMN3299), rad3 Δ crb2 Δ (TMN3300), crb2 Δ (TMN2941), crb2 Δ hta1 hta2 (TMN3297), chk1 Δ (TMN2943), chk1 Δ hta1 hta2 (TMN3298), crb2 Δ chk1 Δ (TMN3305), crb2 Δ chk1 Δ hta1 hta2 (TMN3308), rqh1 Δ (TMN3301), rqh1 Δ hta1 hta2 (TMN3302), crb2 Δ rqh1 Δ (TMN3303), crb2 Δ rqh1 Δ hta1 hta2 (TMN3307), chk1 Δ rqh1 Δ (TMN3304), chk1 Δ rqh1 Δ hta1 hta2 (TMN3422), and crb2 Δ chk1 Δ rqh1 Δ (TMN3306). Error bars indicate standard deviations from at least three experiments. (E) Model depicting the γ-H2A-dependent modulation of Crb2 functions involving Chk1 and Rqh1, based on genetic interactions of IR sensitivity. (F) Model for the mammalian γ-H2AX-dependent DNA damage response. A speculative negative role of the γ-H2AX in DNA repair is indicated as a gray inhibitory sign covered with question marks.

    Article Snippet: For visualization of phosphorylated histone H2A by indirect immunofluorescence microscopy, cells were fixed with 3.7% formaldehyde and processed as previously described ( ) using anti-phospho-H2AX (serine-139) polyclonal immunoglobulin G (IgG; Upstate Biotechnology) and Alexa Fluor 488 goat anti-rabbit IgG (Molecular Probes) as primary and secondary antibodies, respectively.

    Techniques:

    Fission yeast histone H2A is phosphorylated in response to IR. (A) S. pombe histone H2A genes and H2A-AQE mutations. (Top) Organization of histone H2A genes ( hta1 + and hta2 + ). hta1 + is paired to the histone H2B gene ( htb1 + ). AQE mutations are indicated (S129A and S128A). (Bottom) Alignment of histone H2A C-tail sequences from human (H2AX), mouse (H2AX), Tetrahymena thermophila (H2A.1), S. cerevisiae (H2A.1 and H2A.2), and S. pombe (H2A.1 and H2A.2). Conserved amino acids are shaded, and the serine in the SQE motif is indicated with an arrow. (B) H2A phosphorylation detected by Western blotting. Histone-enriched cell extracts were prepared from unirradiated (−) and irradiated (+) S. pombe ( S.p. ) or S. cerevisiae ( S.c. ) cells and separated by sodium dodecyl sulfate-13.5% polyacrylamide gel electrophoresis. γ-H2A signals were detected using the budding yeast γ-H2A peptide antibody (right). The same protein extracts used in the Western blot assay were loaded on another gel and visualized by Coomassie blue staining (left). (C) H2A phosphorylation detected by immunofluorescence microscopy. Unirradiated (-IR) and irradiated (+IR; 192 Gy) S. pombe cells were fixed with formaldehyde immediately (less than 5 min) after irradiation and processed for indirect immunofluorescence microscopy using mammalian γ-H2AX peptide antibody. (D and E) The percentages of cells with γ-H2A signal were determined from three independent experiments with at least 500 cells counted microscopically for each IR dose or each time after IR. Error bars indicate standard deviations. Strains used were wild type (TMN3289), hta1-S129A hta2-S128A (TMN3291), rad3 Δ tel1 Δ (TMN2978), rad3 Δ (TMN2947), tel1 Δ (TMN2967), and rhp51 Δ (PS2383).

    Journal: Molecular and Cellular Biology

    Article Title: Histone H2A Phosphorylation Controls Crb2 Recruitment at DNA Breaks, Maintains Checkpoint Arrest, and Influences DNA Repair in Fission Yeast †

    doi: 10.1128/MCB.24.14.6215-6230.2004

    Figure Lengend Snippet: Fission yeast histone H2A is phosphorylated in response to IR. (A) S. pombe histone H2A genes and H2A-AQE mutations. (Top) Organization of histone H2A genes ( hta1 + and hta2 + ). hta1 + is paired to the histone H2B gene ( htb1 + ). AQE mutations are indicated (S129A and S128A). (Bottom) Alignment of histone H2A C-tail sequences from human (H2AX), mouse (H2AX), Tetrahymena thermophila (H2A.1), S. cerevisiae (H2A.1 and H2A.2), and S. pombe (H2A.1 and H2A.2). Conserved amino acids are shaded, and the serine in the SQE motif is indicated with an arrow. (B) H2A phosphorylation detected by Western blotting. Histone-enriched cell extracts were prepared from unirradiated (−) and irradiated (+) S. pombe ( S.p. ) or S. cerevisiae ( S.c. ) cells and separated by sodium dodecyl sulfate-13.5% polyacrylamide gel electrophoresis. γ-H2A signals were detected using the budding yeast γ-H2A peptide antibody (right). The same protein extracts used in the Western blot assay were loaded on another gel and visualized by Coomassie blue staining (left). (C) H2A phosphorylation detected by immunofluorescence microscopy. Unirradiated (-IR) and irradiated (+IR; 192 Gy) S. pombe cells were fixed with formaldehyde immediately (less than 5 min) after irradiation and processed for indirect immunofluorescence microscopy using mammalian γ-H2AX peptide antibody. (D and E) The percentages of cells with γ-H2A signal were determined from three independent experiments with at least 500 cells counted microscopically for each IR dose or each time after IR. Error bars indicate standard deviations. Strains used were wild type (TMN3289), hta1-S129A hta2-S128A (TMN3291), rad3 Δ tel1 Δ (TMN2978), rad3 Δ (TMN2947), tel1 Δ (TMN2967), and rhp51 Δ (PS2383).

    Article Snippet: For visualization of phosphorylated histone H2A by indirect immunofluorescence microscopy, cells were fixed with 3.7% formaldehyde and processed as previously described ( ) using anti-phospho-H2AX (serine-139) polyclonal immunoglobulin G (IgG; Upstate Biotechnology) and Alexa Fluor 488 goat anti-rabbit IgG (Molecular Probes) as primary and secondary antibodies, respectively.

    Techniques: Western Blot, Irradiation, Polyacrylamide Gel Electrophoresis, Staining, Immunofluorescence, Microscopy

    Genetic interactions of histone H2A-AQE mutations with mutations in DNA repair genes. (A to D) Survival of cells carrying various combinations of mutations in H2A ( hta1-S129A and hta2-S128A , indicated as hta1 hta2 ) and DNA repair ( rad22 Δ, rhp51 Δ, rad32 Δ, rqh1 Δ, pku70 Δ, and lig4 Δ) genes after exposure to the indicated doses of IR. For data in panels C and D, G 1 indicates that cells were arrested in G 1 by nitrogen starvation for 48 h prior to IR treatment (see Materials and Methods), while G 2 indicates exponentially growing cells. Strains used were rad22 Δ (TMN3317), rad22 Δ hta1 hta2 (TMN3318), rhp51 Δ (TMN3319), rhp51 Δ hta1 hta2 (TMN3320), rqh1 Δ (TMN3301), rqh1 Δ hta1 hta2 (TMN3302), rad32 Δ (TMN2800), rad32 Δ hta1 hta2 (TMN3321), wild type (TMN2665), hta1 hta2 (TMN3293), pku70 Δ (TMN3001), pku70 Δ hta1 hta2 (TMN3322), lig4 Δ (PS2818), lig4 Δ hta1 hta2 (TMN3323), rhp51 Δ pku70 Δ (TMN3419), and rhp51 Δ pku70 Δ hta1 hta2 (TMN3420). Error bars indicate standard deviations from at least three experiments. (E) A plasmid repair assay to measure NHEJ efficiency was carried out for the indicated strains. Relative colony numbers obtained from transformation with cut versus uncut plasmid are plotted. Strains used were wild type (TMN2663), hta1 hta2 (TMN3292), pku70 Δ (TMN3001), pku70 Δ hta1 hta2 (TMN3322), lig4 Δ (PS2818), and lig4 Δ hta1 hta2 (TMN3323). Error bars indicate standard deviations from at least three experiments. (F) Generation times for indicated strains, determined from two independent experiments. Error bars indicate differences between two experiments. Strains used were the same as those used for panels A and B, except for wild-type (TMN3296), hta1 hta2 (TMN3291), rad22 Δ (TMN3460), and rad32 Δ (TMN3461) strains.

    Journal: Molecular and Cellular Biology

    Article Title: Histone H2A Phosphorylation Controls Crb2 Recruitment at DNA Breaks, Maintains Checkpoint Arrest, and Influences DNA Repair in Fission Yeast †

    doi: 10.1128/MCB.24.14.6215-6230.2004

    Figure Lengend Snippet: Genetic interactions of histone H2A-AQE mutations with mutations in DNA repair genes. (A to D) Survival of cells carrying various combinations of mutations in H2A ( hta1-S129A and hta2-S128A , indicated as hta1 hta2 ) and DNA repair ( rad22 Δ, rhp51 Δ, rad32 Δ, rqh1 Δ, pku70 Δ, and lig4 Δ) genes after exposure to the indicated doses of IR. For data in panels C and D, G 1 indicates that cells were arrested in G 1 by nitrogen starvation for 48 h prior to IR treatment (see Materials and Methods), while G 2 indicates exponentially growing cells. Strains used were rad22 Δ (TMN3317), rad22 Δ hta1 hta2 (TMN3318), rhp51 Δ (TMN3319), rhp51 Δ hta1 hta2 (TMN3320), rqh1 Δ (TMN3301), rqh1 Δ hta1 hta2 (TMN3302), rad32 Δ (TMN2800), rad32 Δ hta1 hta2 (TMN3321), wild type (TMN2665), hta1 hta2 (TMN3293), pku70 Δ (TMN3001), pku70 Δ hta1 hta2 (TMN3322), lig4 Δ (PS2818), lig4 Δ hta1 hta2 (TMN3323), rhp51 Δ pku70 Δ (TMN3419), and rhp51 Δ pku70 Δ hta1 hta2 (TMN3420). Error bars indicate standard deviations from at least three experiments. (E) A plasmid repair assay to measure NHEJ efficiency was carried out for the indicated strains. Relative colony numbers obtained from transformation with cut versus uncut plasmid are plotted. Strains used were wild type (TMN2663), hta1 hta2 (TMN3292), pku70 Δ (TMN3001), pku70 Δ hta1 hta2 (TMN3322), lig4 Δ (PS2818), and lig4 Δ hta1 hta2 (TMN3323). Error bars indicate standard deviations from at least three experiments. (F) Generation times for indicated strains, determined from two independent experiments. Error bars indicate differences between two experiments. Strains used were the same as those used for panels A and B, except for wild-type (TMN3296), hta1 hta2 (TMN3291), rad22 Δ (TMN3460), and rad32 Δ (TMN3461) strains.

    Article Snippet: For visualization of phosphorylated histone H2A by indirect immunofluorescence microscopy, cells were fixed with 3.7% formaldehyde and processed as previously described ( ) using anti-phospho-H2AX (serine-139) polyclonal immunoglobulin G (IgG; Upstate Biotechnology) and Alexa Fluor 488 goat anti-rabbit IgG (Molecular Probes) as primary and secondary antibodies, respectively.

    Techniques: Plasmid Preparation, Non-Homologous End Joining, Transformation Assay

    DNA repair rate for H2A-AQE mutant cells is essentially the same as for wild-type cells. (A) DNA repair rates of wild-type and hta1-S129A hta2-S128A cells after exposure to IR (192 Gy) were monitored by visualizing reappearance of intact chromosomes after the indicated times by PFGE. Strains used were wild type (TMN2665) and hta1 hta2 (TMN3293). (B) IRIF formation of Rad22 is not affected by H2A-AQE mutations. Time-dependent changes in Rad22-YFP foci after 36-Gy IR for YFP-rad22 (TMN3333) and YFP-rad22 hta1-S129 hta2-S128A (TMN3334) cells are plotted on the right. Representative changes in rad22-YFP localization patterns in response to IR (45 min past IR) are shown in the left panels.

    Journal: Molecular and Cellular Biology

    Article Title: Histone H2A Phosphorylation Controls Crb2 Recruitment at DNA Breaks, Maintains Checkpoint Arrest, and Influences DNA Repair in Fission Yeast †

    doi: 10.1128/MCB.24.14.6215-6230.2004

    Figure Lengend Snippet: DNA repair rate for H2A-AQE mutant cells is essentially the same as for wild-type cells. (A) DNA repair rates of wild-type and hta1-S129A hta2-S128A cells after exposure to IR (192 Gy) were monitored by visualizing reappearance of intact chromosomes after the indicated times by PFGE. Strains used were wild type (TMN2665) and hta1 hta2 (TMN3293). (B) IRIF formation of Rad22 is not affected by H2A-AQE mutations. Time-dependent changes in Rad22-YFP foci after 36-Gy IR for YFP-rad22 (TMN3333) and YFP-rad22 hta1-S129 hta2-S128A (TMN3334) cells are plotted on the right. Representative changes in rad22-YFP localization patterns in response to IR (45 min past IR) are shown in the left panels.

    Article Snippet: For visualization of phosphorylated histone H2A by indirect immunofluorescence microscopy, cells were fixed with 3.7% formaldehyde and processed as previously described ( ) using anti-phospho-H2AX (serine-139) polyclonal immunoglobulin G (IgG; Upstate Biotechnology) and Alexa Fluor 488 goat anti-rabbit IgG (Molecular Probes) as primary and secondary antibodies, respectively.

    Techniques: Mutagenesis

    H2A-AQE mutant cells show a defect in checkpoint maintenance under continuous exposure to bleomycin. (A) Wild-type, H2A-AQE ( hta1-S129A hta2-S128A ), or tel1 Δ cells in either a rad3 + or rad3 ts ( rad3 ts -V2217A ) background were incubated with bleomycin for 2 h at 25°C, shifted to 35°C, fixed with glutaraldehyde, and stained with calcofluor, and the percentage of septated cells was counted for each time point. The micrographic pictures on the right are representative cells from the experiments graphed on the left. Cells were fixed and stained with 4,6-diamidino-2-phenylindole (DAPI) to visualize DNA and septa. Strains used were wild type (TMN2665), hta1 hta2 (TMN3293), rad3 ts (TMN3313), and rad3 ts hta1 hta2 (TMN3314). (B) Cells with integrated chk1-myc in either wild-type (TMN3309), hta1-S129A hta2-S128A (TMN3310), rad3 ts (TMN3315), or rad3 ts hta1-S129A hta2-S128A (TMN3316) backgrounds were incubated with bleomycin for 2 h at 25°C and then shifted to 35°C. Aliquots were taken at the indicated time points after the temperature shift, and cell extracts were prepared. Chk1-myc was visualized by Western blotting. Arrows indicate phosphorylated forms of Chk1.

    Journal: Molecular and Cellular Biology

    Article Title: Histone H2A Phosphorylation Controls Crb2 Recruitment at DNA Breaks, Maintains Checkpoint Arrest, and Influences DNA Repair in Fission Yeast †

    doi: 10.1128/MCB.24.14.6215-6230.2004

    Figure Lengend Snippet: H2A-AQE mutant cells show a defect in checkpoint maintenance under continuous exposure to bleomycin. (A) Wild-type, H2A-AQE ( hta1-S129A hta2-S128A ), or tel1 Δ cells in either a rad3 + or rad3 ts ( rad3 ts -V2217A ) background were incubated with bleomycin for 2 h at 25°C, shifted to 35°C, fixed with glutaraldehyde, and stained with calcofluor, and the percentage of septated cells was counted for each time point. The micrographic pictures on the right are representative cells from the experiments graphed on the left. Cells were fixed and stained with 4,6-diamidino-2-phenylindole (DAPI) to visualize DNA and septa. Strains used were wild type (TMN2665), hta1 hta2 (TMN3293), rad3 ts (TMN3313), and rad3 ts hta1 hta2 (TMN3314). (B) Cells with integrated chk1-myc in either wild-type (TMN3309), hta1-S129A hta2-S128A (TMN3310), rad3 ts (TMN3315), or rad3 ts hta1-S129A hta2-S128A (TMN3316) backgrounds were incubated with bleomycin for 2 h at 25°C and then shifted to 35°C. Aliquots were taken at the indicated time points after the temperature shift, and cell extracts were prepared. Chk1-myc was visualized by Western blotting. Arrows indicate phosphorylated forms of Chk1.

    Article Snippet: For visualization of phosphorylated histone H2A by indirect immunofluorescence microscopy, cells were fixed with 3.7% formaldehyde and processed as previously described ( ) using anti-phospho-H2AX (serine-139) polyclonal immunoglobulin G (IgG; Upstate Biotechnology) and Alexa Fluor 488 goat anti-rabbit IgG (Molecular Probes) as primary and secondary antibodies, respectively.

    Techniques: Mutagenesis, Incubation, Staining, Western Blot

    BRE overexpression affects IR-induced CDC25A degradation. a DNA synthesis in ES cells after different doses of IR. b Western blot analysis of CDC25A and Tyr15 phosphorylation of CDK1 (p-CDK1) at different times after exposing the cells to 6 Gy IR in Brca2 CKO/KO ;MSCV-BRE cells compared to Brca2 CKO/KO cells. GAPDH was used as a loading control. Numbers below indicate the lane numbers. The quantification of CDC25A (middle panel) and p-CDK1 (lower panel) band intensity for each cell line is shown as histogram. Relative band intensities were calculated by dividing GAPDH-normalized CDC25A/p-CDK1 intensity at particular time point with GAPDH-normalized CDC25A/p-CDK1 intensity of corresponding untreated cells. c MCF7 cells with inducible HA-BRE expression cassette were transfected with CRE plasmid and subjected to 6 Gy IR after 48 h of transfection. Western blots of endogenous CDC25A in presence (lanes 4–6) and absence (lanes 1–3) of HA-BRE expression at different times after irradiation is shown in the upper panel. Lower panel shows the quantification of CDC25A band intensity normalized against GAPDH. d Immunoblot showing CDC25A degradation after IR treatment in MCF7 cells transfected either with non-specific (NS) siRNA or two different siRNAs against BRE (#1 and #2). Histone H3 was used as a loading control. Relative CDC25A band intensities were calculated by dividing GAPDH-normalized CDC25A intensity at a particular time point with GAPDH-normalized CDC25A intensity of untreated (0 h IR) cells of corresponding siRNA treatment. e Western blot showing IR-induced CDC25A degradation in MCF7 cells with stably integrated CRE inducible HA-BRE expression cassette after transfection either with non-specific (NS) siRNA or two different siRNAs against MERIT40 (#1 and #2) along with CRE -expressing plasmid. Non-specific (NS) siRNA transfection was used as control. Relative abundance of CDC25A at a particular time point was measured by comparing GAPDH-normalized band intensities at that point divided by GAPDH-normalized CDC25A intensity of untreated (0 h IR) cells of corresponding treatment. All histograms show the average of three independent experiments and error bars represent s.d. P values were calculated using paired two-tailed t

    Journal: Nature Communications

    Article Title: BRE/BRCC45 regulates CDC25A stability by recruiting USP7 in response to DNA damage

    doi: 10.1038/s41467-018-03020-6

    Figure Lengend Snippet: BRE overexpression affects IR-induced CDC25A degradation. a DNA synthesis in ES cells after different doses of IR. b Western blot analysis of CDC25A and Tyr15 phosphorylation of CDK1 (p-CDK1) at different times after exposing the cells to 6 Gy IR in Brca2 CKO/KO ;MSCV-BRE cells compared to Brca2 CKO/KO cells. GAPDH was used as a loading control. Numbers below indicate the lane numbers. The quantification of CDC25A (middle panel) and p-CDK1 (lower panel) band intensity for each cell line is shown as histogram. Relative band intensities were calculated by dividing GAPDH-normalized CDC25A/p-CDK1 intensity at particular time point with GAPDH-normalized CDC25A/p-CDK1 intensity of corresponding untreated cells. c MCF7 cells with inducible HA-BRE expression cassette were transfected with CRE plasmid and subjected to 6 Gy IR after 48 h of transfection. Western blots of endogenous CDC25A in presence (lanes 4–6) and absence (lanes 1–3) of HA-BRE expression at different times after irradiation is shown in the upper panel. Lower panel shows the quantification of CDC25A band intensity normalized against GAPDH. d Immunoblot showing CDC25A degradation after IR treatment in MCF7 cells transfected either with non-specific (NS) siRNA or two different siRNAs against BRE (#1 and #2). Histone H3 was used as a loading control. Relative CDC25A band intensities were calculated by dividing GAPDH-normalized CDC25A intensity at a particular time point with GAPDH-normalized CDC25A intensity of untreated (0 h IR) cells of corresponding siRNA treatment. e Western blot showing IR-induced CDC25A degradation in MCF7 cells with stably integrated CRE inducible HA-BRE expression cassette after transfection either with non-specific (NS) siRNA or two different siRNAs against MERIT40 (#1 and #2) along with CRE -expressing plasmid. Non-specific (NS) siRNA transfection was used as control. Relative abundance of CDC25A at a particular time point was measured by comparing GAPDH-normalized band intensities at that point divided by GAPDH-normalized CDC25A intensity of untreated (0 h IR) cells of corresponding treatment. All histograms show the average of three independent experiments and error bars represent s.d. P values were calculated using paired two-tailed t

    Article Snippet: The antibodies used for co-immunoprecipitation, immunofluorescence, and western blots were anti-BRE (C-16: sc-48847 and sc-376453; Santa Cruz Biotech, 1:500 dilution for western blots), anti-BRCC36 (ab62075; Abcam and sc131122; Santa Cruz Biotech, 1:1000 dilution for western blots), anti-CDC25A (F-6: sc-7389; Santa Cruz Biotech, 1:200 dilution), anti-FLAG (F7425 and F3165; Sigma-Aldrich, 1:2000 dilution for western blot), anti-HA (12013819001; Roche, 1:2000 dilution), anti-USP7 (H-200: sc-30164 and H12: sc-137008; Santa Cruz Biotech and ab4080; Abcam, 1:1000 dilution for western blot), anti-β TRCP (ab118006; Abcam, 1:500 dilution), anti-DUB3 (ab174914; Abcam and H00377630-M01; Abnova, 1:1000 dilution for western blot), anti-RAP80 (A300-764A-M; Bethyl laboratories, 1:500 dilution for western blot), K-48 linkage specific antibody (#4289S; Cell Signaling, 1:500 dilution), K-63 linkage specific antibody (#5621S; Cell Signaling, 1:500 dilution), anti-histone H3 (#9715; Cell Signaling, 1:5000 dilution), anti-HDM2 (sc-56154; Santa Cruz Biotech, 1:500 dilution), anti GAPDH (2118S; Cell Signaling, 1:40,000 dilution), anti-ACTIN (C2: sc-8432; Santa Cruz Biotech, 1:40,000 dilution), anti-phosphorylated CHK1 (12302; Cell Signaling, 1:500 dilution), anti-phosphorylated CDK1 (#9111; Cell Signaling, 1:1000 dilution), anti-p53 (#2524; Cell Signaling, 1:5000 dilution), anti-RAD51 (PC130; Calbiochem, 1:2000 for western and 1:250 for Immunofluorescence), anti-γH2AX (05-636; Upstate), anti-ubiquitin (P4D1: sc-8017; Santa Cruz Biotech, 1:1000 dilution), anti-Myc (631206, Clontech and 2272S; Cell Signaling, 1:1000 dilution), anti-MERIT40 (#12711; Cell Signaling, 1:1000 dilution), and anti-BRCA2 (A303-434A-T; Bethyl Laboratories, 1:2000 dilution).

    Techniques: Over Expression, DNA Synthesis, Western Blot, Expressing, Transfection, Plasmid Preparation, Irradiation, Stable Transfection, Two Tailed Test

    Enhancer profiling of maEpiSCs. ( A ) H3K27ac densities shown using a boxplot and ( B ) scatter plot (normalized tag density; log2 RPBM) at EpiSC-regions in conventional EpiSCs, maEpiSCs, MEFs, and ESCs. ( C ) H3K27ac densities shown using a boxplot at ESC-defined super-enhancers. ( D ) Analysis of H3K27ac levels in maEpiSCs, conventional EpiSCs, ESCs and MEFs using principal component analysis (PCA). ( E ) H3K27ac densities shown in heat maps at EpiSC-defined peaks. ( F ) Overlap between H3K27ac in maEpiSCs and ES cells (top left), maEpiSCs and conventional EpiSCs (middle left), and maEpiSCs and MEFs (bottom left) shown using Venn diagrams. ( G ) Venn diagrams of H3K4me2 levels between maEpiSCs and ES cells (top right), maEpiSCs and conventional EpiSCs (middle right), and maEpiSCs and MEFs (bottom right). ( H ) H3K4me2 densities shown using a boxplot and ( I ) scatter plots at EpiSC-defined regions. ( J ) H3K4me2 density shown using a boxplot at ESC-defined super-enhancers. ( K ) PCA of H3K4me2 levels between maEpiSCs, conventional EpiSCs, ES cells and MEFs. ( L ) Heat maps of H3K4me2 densities at transcriptional start site (TSS) regions. ( M ) Custom UCSC views of H3K4me2 and H3K27ac levels in conventional EpiSCs, maEpiSCs, MEFs, and ESCs.

    Journal: Scientific Reports

    Article Title: Culture of haploid blastocysts in FGF4 favors the derivation of epiblast stem cells with a primed epigenetic and transcriptional landscape

    doi: 10.1038/s41598-018-29074-6

    Figure Lengend Snippet: Enhancer profiling of maEpiSCs. ( A ) H3K27ac densities shown using a boxplot and ( B ) scatter plot (normalized tag density; log2 RPBM) at EpiSC-regions in conventional EpiSCs, maEpiSCs, MEFs, and ESCs. ( C ) H3K27ac densities shown using a boxplot at ESC-defined super-enhancers. ( D ) Analysis of H3K27ac levels in maEpiSCs, conventional EpiSCs, ESCs and MEFs using principal component analysis (PCA). ( E ) H3K27ac densities shown in heat maps at EpiSC-defined peaks. ( F ) Overlap between H3K27ac in maEpiSCs and ES cells (top left), maEpiSCs and conventional EpiSCs (middle left), and maEpiSCs and MEFs (bottom left) shown using Venn diagrams. ( G ) Venn diagrams of H3K4me2 levels between maEpiSCs and ES cells (top right), maEpiSCs and conventional EpiSCs (middle right), and maEpiSCs and MEFs (bottom right). ( H ) H3K4me2 densities shown using a boxplot and ( I ) scatter plots at EpiSC-defined regions. ( J ) H3K4me2 density shown using a boxplot at ESC-defined super-enhancers. ( K ) PCA of H3K4me2 levels between maEpiSCs, conventional EpiSCs, ES cells and MEFs. ( L ) Heat maps of H3K4me2 densities at transcriptional start site (TSS) regions. ( M ) Custom UCSC views of H3K4me2 and H3K27ac levels in conventional EpiSCs, maEpiSCs, MEFs, and ESCs.

    Article Snippet: Briefly, the H3K4me3 antibody (17–614) was obtained from Millipore, while the H3K27me3 (ab6002), H3K27ac (ab4729), and H3K4me2 (ab32356) antibodies were obtained from Abcam.

    Techniques:

    Characterization of micronuclei in t e × l s hybrid embryos and link to embryo death a, Disrupted micronuclei envelopes in t e × l s hybrid embryos. Whole mount embryo immunofluorescence was performed in t e × l s hybrid embryos using the YO-PRO DNA dye (top) and anti-Lamin B1 antibody (middle), corresponding channels are shown in green and magenta, respectively. The merged images are shown below. 25 micronuclei within 5 different embryos were analyzed. Intact (left) and disrupted (right) envelopes were observed in all analyzed embryos. Scale bar is 10 μm. b, DNA damage in t e × l s hybrid embryo micronuclei. Whole mount embryo immunofluorescence was performed in t e × l s hybrid embryos using anti-histone H3 (top) and anti-γH2A.X (middle) antibodies, corresponding channels are shown in green and magenta, respectively. The merge images are shown below. 21 micronuclei within different 6 embryos were analyzed. Micronuclei with undamaged (left; negative γH2A.X signal) and damaged (right; positive γH2A.X signal) DNA were observed in all analyzed embryos. Zoomed images of micronuclei are shown on the right of each image. Scale bar is 10 μm. c, TUNEL assay in apoptotic X. tropicalis and t e × l s hybrid embryos. X. tropicalis (left), X. tropicalis treated with cycloheximide (middle left) or hydroxyurea (middle right) as indicated, and t e × l s hybrid (right) embryos were prepared for TUNEL assay 5 hpf (equivalent stage 9; top), 7 hpf (equivalent stage 10; middle) and 9.5 hpf (equivalent stage 10.5; bottom). Identical results were obtained over 3 different experiments. Representative images are shown and were taken under identical conditions.

    Journal: Nature

    Article Title: Paternal chromosome loss and metabolic crisis contribute to hybrid inviability in Xenopus

    doi: 10.1038/nature25188

    Figure Lengend Snippet: Characterization of micronuclei in t e × l s hybrid embryos and link to embryo death a, Disrupted micronuclei envelopes in t e × l s hybrid embryos. Whole mount embryo immunofluorescence was performed in t e × l s hybrid embryos using the YO-PRO DNA dye (top) and anti-Lamin B1 antibody (middle), corresponding channels are shown in green and magenta, respectively. The merged images are shown below. 25 micronuclei within 5 different embryos were analyzed. Intact (left) and disrupted (right) envelopes were observed in all analyzed embryos. Scale bar is 10 μm. b, DNA damage in t e × l s hybrid embryo micronuclei. Whole mount embryo immunofluorescence was performed in t e × l s hybrid embryos using anti-histone H3 (top) and anti-γH2A.X (middle) antibodies, corresponding channels are shown in green and magenta, respectively. The merge images are shown below. 21 micronuclei within different 6 embryos were analyzed. Micronuclei with undamaged (left; negative γH2A.X signal) and damaged (right; positive γH2A.X signal) DNA were observed in all analyzed embryos. Zoomed images of micronuclei are shown on the right of each image. Scale bar is 10 μm. c, TUNEL assay in apoptotic X. tropicalis and t e × l s hybrid embryos. X. tropicalis (left), X. tropicalis treated with cycloheximide (middle left) or hydroxyurea (middle right) as indicated, and t e × l s hybrid (right) embryos were prepared for TUNEL assay 5 hpf (equivalent stage 9; top), 7 hpf (equivalent stage 10; middle) and 9.5 hpf (equivalent stage 10.5; bottom). Identical results were obtained over 3 different experiments. Representative images are shown and were taken under identical conditions.

    Article Snippet: We used different combinations of the following antibodies: 1:500 mouse anti-beta tubulin (E7; Developmental Studies Hybridoma Bank, Iowa City, IA), 1:500 rabbit anti-histone H3 (ab1791; Abcam, Cambridge, MA), 1:500 rabbit anti-lamin B1 (ab16048; Abcam, Cambridge, MA), 1:500 mouse anti-phospho-histone H2A.X (05-636; EMD Millipore, Merck KGaA, Darmstadt, Germany).

    Techniques: Immunofluorescence, TUNEL Assay

    Occurrence of micronuclei, role of ploidy and spindle architecture a, Micronuclei in t e × l s hybrid embryos at various developmental stages. Whole mount embryo immunofluorescence was performed in t e × l s hybrid embryos using anti-histone H3 antibody at stages 4, 6, 7, 8 and 9 and quantified in b. Scale bar is 10 μm. b, Quantification of micronuclei in t e × l s hybrid embryos. The percentage of micronuclei was calculated as the number of micronuclei in the imaged portion of the embryo divided by the total number of nuclei in the same imaged portion. The average percentage for multiple embryos at stage 4 (n = 18 t e × l s hybrid embryos (individual dots) with a total of 63 nuclei), stage 6 (n = 17/115), stage 7 (n = 9/322), stage 8 (n = 8/1119) and stage 9 (n = 3/2004) from 3 independent experiments, is shown as thick line. Gray boxes indicate a 1 standard error of the mean. Control X. tropicalis embryos from the same mothers were analyzed but no micronuclei were observed at any stages. c, Nuclear size in X. tropicalis embryos with varying ploidy. Nuclear size relative to cell size (diameters in μm) is plotted for triploid ( tt e × t s ; dark grey, n = 175 nuclei from 6 embryos), diploid ( X. tropicalis , t e × t s ; blue, n = 453/9) and haploid ( t e ×[ t s ]; light grey, n = 346/16) embryos. Each dot indicates an individual data point and the solid lines indicate a linear fit. d, X. tropicalis embryos with varying ploidy at tailbud stage. Images of triploid ( tt e × t s ; left) and haploid ( t e ×[ t s ]; right) tailbuds were taken under identical conditions. Similar observations were over 3 independent experiments. e, Size and microtubule distribution in X. tropicalis spindles assembled from different embryo nuclei DNA (n = 147, 103, and 156 spindles quantified for X. tropicalis , l e × t s hybrids, and X. laevis embryo nuclei, respectively, from 3 different egg extracts). Spindle length (left) and width (middle) were normalized to the X. tropicalis control, averages are shown as thick black lines and the gray boxes indicate 1 standard deviation. 95% confidence intervals for lengths are 1±0.02 for t e × t s , 1.05±0.03 for l e × t s , and 1.03±0.02 for l e × l s and for widths are 1±0.04, 1.3±0.07, and 1.4±0.04. Line scans of rhodamine-tubulin signal along spindle length were taken (right). Spindle lengths were normalized to 100% and tubulin intensities were normalized within datasets. The average intensities are plotted for the three spindle types, error bars indicate standard deviation and colors are as in . Figure 2a

    Journal: Nature

    Article Title: Paternal chromosome loss and metabolic crisis contribute to hybrid inviability in Xenopus

    doi: 10.1038/nature25188

    Figure Lengend Snippet: Occurrence of micronuclei, role of ploidy and spindle architecture a, Micronuclei in t e × l s hybrid embryos at various developmental stages. Whole mount embryo immunofluorescence was performed in t e × l s hybrid embryos using anti-histone H3 antibody at stages 4, 6, 7, 8 and 9 and quantified in b. Scale bar is 10 μm. b, Quantification of micronuclei in t e × l s hybrid embryos. The percentage of micronuclei was calculated as the number of micronuclei in the imaged portion of the embryo divided by the total number of nuclei in the same imaged portion. The average percentage for multiple embryos at stage 4 (n = 18 t e × l s hybrid embryos (individual dots) with a total of 63 nuclei), stage 6 (n = 17/115), stage 7 (n = 9/322), stage 8 (n = 8/1119) and stage 9 (n = 3/2004) from 3 independent experiments, is shown as thick line. Gray boxes indicate a 1 standard error of the mean. Control X. tropicalis embryos from the same mothers were analyzed but no micronuclei were observed at any stages. c, Nuclear size in X. tropicalis embryos with varying ploidy. Nuclear size relative to cell size (diameters in μm) is plotted for triploid ( tt e × t s ; dark grey, n = 175 nuclei from 6 embryos), diploid ( X. tropicalis , t e × t s ; blue, n = 453/9) and haploid ( t e ×[ t s ]; light grey, n = 346/16) embryos. Each dot indicates an individual data point and the solid lines indicate a linear fit. d, X. tropicalis embryos with varying ploidy at tailbud stage. Images of triploid ( tt e × t s ; left) and haploid ( t e ×[ t s ]; right) tailbuds were taken under identical conditions. Similar observations were over 3 independent experiments. e, Size and microtubule distribution in X. tropicalis spindles assembled from different embryo nuclei DNA (n = 147, 103, and 156 spindles quantified for X. tropicalis , l e × t s hybrids, and X. laevis embryo nuclei, respectively, from 3 different egg extracts). Spindle length (left) and width (middle) were normalized to the X. tropicalis control, averages are shown as thick black lines and the gray boxes indicate 1 standard deviation. 95% confidence intervals for lengths are 1±0.02 for t e × t s , 1.05±0.03 for l e × t s , and 1.03±0.02 for l e × l s and for widths are 1±0.04, 1.3±0.07, and 1.4±0.04. Line scans of rhodamine-tubulin signal along spindle length were taken (right). Spindle lengths were normalized to 100% and tubulin intensities were normalized within datasets. The average intensities are plotted for the three spindle types, error bars indicate standard deviation and colors are as in . Figure 2a

    Article Snippet: We used different combinations of the following antibodies: 1:500 mouse anti-beta tubulin (E7; Developmental Studies Hybridoma Bank, Iowa City, IA), 1:500 rabbit anti-histone H3 (ab1791; Abcam, Cambridge, MA), 1:500 rabbit anti-lamin B1 (ab16048; Abcam, Cambridge, MA), 1:500 mouse anti-phospho-histone H2A.X (05-636; EMD Millipore, Merck KGaA, Darmstadt, Germany).

    Techniques: Immunofluorescence, Standard Deviation

    RELB-P53 suppresses endothelial growth in the absence of PKM2. a EdU and phospho-Histone H3 positive cell numbers in control, RELB KD , PKM2 KD and PKM2 KD /RELB KD ECs ( n = 6). b Western blot analysis of RELB, P53, P21 and PKM2 expression in control, RELB KD , PKM2 KD and PKM2 KD /RELB KD ECs. c EdU and phospho-Histone H3 positive cell numbers in control, P53 KD , PKM2 KD and PKM2 KD /P53 KD ECs ( n = 12). d Western blot analysis of RELB, P53, P21 and PKM2 expression in control, P53 KD , PKM2 KD and PKM2 KD /P53 KD ECs. e mRNA expression levels of key enzymes for pyrimidine synthesis in control, P53 KD , PKM2 KD and PKM2 KD /P53 KD ECs (RNA-seq analysis, n = 3). f Relative N -carbamoyl aspartate levels, UTP levels, incorporation of [U-13C 6 ]-glucose into m+3 and m+5 UTP, and relative dTTP levels in control, P53 KD , PKM2 KD and PKM2 KD /P53 KD ECs ( n = 3). a , c Data represent means ± s.e.m., e , f data represent means ± s.d., m+ n represents the mass isotopomers for individual metabolites (*** P

    Journal: Nature Communications

    Article Title: Loss of pyruvate kinase M2 limits growth and triggers innate immune signaling in endothelial cells

    doi: 10.1038/s41467-018-06406-8

    Figure Lengend Snippet: RELB-P53 suppresses endothelial growth in the absence of PKM2. a EdU and phospho-Histone H3 positive cell numbers in control, RELB KD , PKM2 KD and PKM2 KD /RELB KD ECs ( n = 6). b Western blot analysis of RELB, P53, P21 and PKM2 expression in control, RELB KD , PKM2 KD and PKM2 KD /RELB KD ECs. c EdU and phospho-Histone H3 positive cell numbers in control, P53 KD , PKM2 KD and PKM2 KD /P53 KD ECs ( n = 12). d Western blot analysis of RELB, P53, P21 and PKM2 expression in control, P53 KD , PKM2 KD and PKM2 KD /P53 KD ECs. e mRNA expression levels of key enzymes for pyrimidine synthesis in control, P53 KD , PKM2 KD and PKM2 KD /P53 KD ECs (RNA-seq analysis, n = 3). f Relative N -carbamoyl aspartate levels, UTP levels, incorporation of [U-13C 6 ]-glucose into m+3 and m+5 UTP, and relative dTTP levels in control, P53 KD , PKM2 KD and PKM2 KD /P53 KD ECs ( n = 3). a , c Data represent means ± s.e.m., e , f data represent means ± s.d., m+ n represents the mass isotopomers for individual metabolites (*** P

    Article Snippet: The following primary antibodies were used: phospho-Histone H3 (Cell Signaling Technology, #9713, 1:500) and J2A (Scicons, 1:200).

    Techniques: Western Blot, Expressing, RNA Sequencing Assay

    miR-222 suppresses BRG1- and STAT-dependent inflammatory gene expression a effect on LPS-induced gene expression. b , ChIP in iBMDMs transduced with overexpression constructs (n=3 independent experiments; p-values from Students t-test for paired values, 2-sided). c-d , Schematic of treatments (c) and genes (d) analyzed in (e-i). e-f transcription factor motifs, and statistically over-represented gene ontology terms (determined by PANTHER) for indicated gene groups (n=103 gene expression values/group). g-h , Transcription factor occupancy (g) and histone modification (h) at promoters, quantified from published ChIP-seq datasets. i , ChIP for STAT2 occupancy in peritoneal macrophages. Values normalized to maximal binding detected for each ChIP (WT, miR-221/222 KO n=4 biologically independent samples; Stat1/2 KO n=2 biologically independent samples. p-values only calculated for WT vs. miR-221/222 KO comparisons by Students t-test, 2-sided, heteroscedastic). j , Model of miR-221/222 effect on chromatin at affected gene promoters. For all bar graphs and dot plots, center represents mean and error bars (if present) the SEM.

    Journal: Nature

    Article Title: Induction of innate immune memory via microRNA targeting of chromatin remodeling factors

    doi: 10.1038/s41586-018-0253-5

    Figure Lengend Snippet: miR-222 suppresses BRG1- and STAT-dependent inflammatory gene expression a effect on LPS-induced gene expression. b , ChIP in iBMDMs transduced with overexpression constructs (n=3 independent experiments; p-values from Students t-test for paired values, 2-sided). c-d , Schematic of treatments (c) and genes (d) analyzed in (e-i). e-f transcription factor motifs, and statistically over-represented gene ontology terms (determined by PANTHER) for indicated gene groups (n=103 gene expression values/group). g-h , Transcription factor occupancy (g) and histone modification (h) at promoters, quantified from published ChIP-seq datasets. i , ChIP for STAT2 occupancy in peritoneal macrophages. Values normalized to maximal binding detected for each ChIP (WT, miR-221/222 KO n=4 biologically independent samples; Stat1/2 KO n=2 biologically independent samples. p-values only calculated for WT vs. miR-221/222 KO comparisons by Students t-test, 2-sided, heteroscedastic). j , Model of miR-221/222 effect on chromatin at affected gene promoters. For all bar graphs and dot plots, center represents mean and error bars (if present) the SEM.

    Article Snippet: Immunoprecipitation was performed using 20 μl magnetic protein A beads and 5 μg anti-acetyl-histone H4 (Lys5; Millipore 07-327), 2 μg Brg1 (H-88; Santa Cruz sc-10768), or 5 μg acetyl-histone H3 (Millipore 06-599) per 50 μl of chromatin in a 500 ul volume.

    Techniques: Expressing, Chromatin Immunoprecipitation, Transduction, Over Expression, Construct, Modification, Binding Assay

    Evidence of miR-222 targeting of Brg1 a , Example of gating that was used to exclude dead cells from flow cytometry analyses in (c), (g), and . b , Example of gating used to distinguish cells with high vs. low levels of IκBα, as analyzed in (c). c , Effect of miRNA overexpression (by viral transduction) on LPS-induced IκBα degradation in iBMDMs, measured by flow cytometry (n=4 independent experiments). d , Sequence and prediction scores of a miR-222 binding site in the Brg1 UTR. e , miR-222 and Brg1 mRNA levels in LPS-stimulated BMDMs (n=3 biologically independent samples). f , Brg1 mRNA levels in resting BMDMs 24 hours after transfection (n=4 biologically independent samples). g , Effect of miRNA overexpression or antagonization (by viral transduction) on BRG1 levels in iBMDMs, observed by flow cytometry. Representative of 4 independent experiments with similar results, quantified in (h). h , Flow cytometry analysis of BRG1 protein levels in transduced iBMDMs (n=4 independent experiments). i , Activity of a luciferase reporter construct in which the luciferase coding sequence is followed by either the complete Brg1 UTR, or a UTR in which the predicted miR-222 binding site has been mutated to the sequence shown in (d) (n=3 independent experiments). j , Quantification of average effect of miR-222 mimic transfection on Brg1 -dependent and –independent LPS-response genes (n=3 biologically independent samples). Two-sided Student’s t-test for heteroscedastic values used to compare ratios (miR-222 overexpression/control) at peak LPS-induced expression times for Brg1 -dependent vs. -independent genes. k-l , ChIP for histone H3 acetylation (k), or histone H4 acetylation (l) after LPS stimulation of iBMDMs transduced with overexpression constructs (k-l tested in same n=3 independent experiments). m , Successful deletion of the miR-222 binding site in the Brg1 UTR in RAW cell clones was confirmed by sequencing genomic DNA of the given cell line. miR-222 binding site is highlighted in yellow. n , Effect of miR-222 overexpression (by oligonucleotide transfection) on LPS-induced gene expression in either a RAW cell line in which the Brg1 ) or a cell line in which the binding site was not targeted for deletion (n=5 independent experiments). For all bar graphs, mean +/− SEM is plotted. ** p

    Journal: Nature

    Article Title: Induction of innate immune memory via microRNA targeting of chromatin remodeling factors

    doi: 10.1038/s41586-018-0253-5

    Figure Lengend Snippet: Evidence of miR-222 targeting of Brg1 a , Example of gating that was used to exclude dead cells from flow cytometry analyses in (c), (g), and . b , Example of gating used to distinguish cells with high vs. low levels of IκBα, as analyzed in (c). c , Effect of miRNA overexpression (by viral transduction) on LPS-induced IκBα degradation in iBMDMs, measured by flow cytometry (n=4 independent experiments). d , Sequence and prediction scores of a miR-222 binding site in the Brg1 UTR. e , miR-222 and Brg1 mRNA levels in LPS-stimulated BMDMs (n=3 biologically independent samples). f , Brg1 mRNA levels in resting BMDMs 24 hours after transfection (n=4 biologically independent samples). g , Effect of miRNA overexpression or antagonization (by viral transduction) on BRG1 levels in iBMDMs, observed by flow cytometry. Representative of 4 independent experiments with similar results, quantified in (h). h , Flow cytometry analysis of BRG1 protein levels in transduced iBMDMs (n=4 independent experiments). i , Activity of a luciferase reporter construct in which the luciferase coding sequence is followed by either the complete Brg1 UTR, or a UTR in which the predicted miR-222 binding site has been mutated to the sequence shown in (d) (n=3 independent experiments). j , Quantification of average effect of miR-222 mimic transfection on Brg1 -dependent and –independent LPS-response genes (n=3 biologically independent samples). Two-sided Student’s t-test for heteroscedastic values used to compare ratios (miR-222 overexpression/control) at peak LPS-induced expression times for Brg1 -dependent vs. -independent genes. k-l , ChIP for histone H3 acetylation (k), or histone H4 acetylation (l) after LPS stimulation of iBMDMs transduced with overexpression constructs (k-l tested in same n=3 independent experiments). m , Successful deletion of the miR-222 binding site in the Brg1 UTR in RAW cell clones was confirmed by sequencing genomic DNA of the given cell line. miR-222 binding site is highlighted in yellow. n , Effect of miR-222 overexpression (by oligonucleotide transfection) on LPS-induced gene expression in either a RAW cell line in which the Brg1 ) or a cell line in which the binding site was not targeted for deletion (n=5 independent experiments). For all bar graphs, mean +/− SEM is plotted. ** p

    Article Snippet: Immunoprecipitation was performed using 20 μl magnetic protein A beads and 5 μg anti-acetyl-histone H4 (Lys5; Millipore 07-327), 2 μg Brg1 (H-88; Santa Cruz sc-10768), or 5 μg acetyl-histone H3 (Millipore 06-599) per 50 μl of chromatin in a 500 ul volume.

    Techniques: Flow Cytometry, Cytometry, Over Expression, Transduction, Sequencing, Binding Assay, Transfection, Activity Assay, Luciferase, Construct, Expressing, Chromatin Immunoprecipitation, Clone Assay

    Piezo + /EP cells are ISC-derived EE precursors with reduced mitotic ability. a. Midguts from flies treated with Bleomycin (10 μg/ml Bleo in 5% sucrose) or the γ-secretase inhibitor DAPT (4 mM DAPT in 5% sucrose). Cells that are positive for both Piezo and Pros are indicated by arrowheads. Note that the majority ( > 95%) of Piezo and Pros double positive cells are also positive for esg, suggesting that these cells are “newborn” EEs that still retain esg-GFP signal. b. Percentage of the newborn EEs (Piezo and Pros double positive cells vs. total Pros + EEs) in fly midguts under control, Bleomycin, and DAPT treatments. Cells within 200 μm X 200 μm areas, n=27 (control), n=25 (Bleo), and n=22 (DAPT), were analyzed. c. Midgut with stem cells labeled by esg-GFP (Green), Piezo + cells labeled by Piezo-Gal4 > nlsRFP (Red), and mitotic cells labeled by anti-phospho-Histone H3 (pH3) (Magenta). pH3 + mitotic cell is indicated by arrowhead. d,e. Representative images of midguts from flies feed on either control (5% sucrose) or Bleomycin (5% sucrose plus 10ug/ml Bleomycin) food. Piezo + EP cells are labeled by Piezo-Gal4 > nlsGFP (Green), mitotic cells are labeled by pH3 staining (Red). Mitotic Piezo + cells are indicated by arrowheads. Since all pH3 + cells are Dl + cells (according to Dl-lacZ labeled midgut), we counted all Piezo negative pH3 + cells as pH3 + ISCs. Under both control (5% sucrose) and damage (5% sucrose + 10 μg/ml Bleomycin) conditions, only around 8–10% of the pH3 + cells are Piezo + (~40% of total Dl + cells), suggesting that Piezo + cells are significantly less mitotically active compared to Piezo- Dl + cells. f. If the pH3 + Piezo + cells are also Pros + . Around 50% of these pH3 + Piezo + cells show low levels of Pros staining. Meanwhile, all the pH3 + Pros + cells are positive for Piezo, suggesting that Piezo + EP cells represent more general EE precursor cells compared to EMCs. Mitotic Piezo + cells are indicated by arrowheads. All experiments were independently repeated at least twice with similar results presented in the figures. g. Random GFP + clones were generated using hsFlp; Ubi-(FRT.Stop)GFP/Piezo-Gal4; UAS-nlsRFP . 3–4 days old flies were heat shocked at 37°C for 30 min once to induce clones in ISCs. Then these flies were kept at 25°C for 2 weeks before analysis. Within each GFP + clone, which is derived from ISCs, there are typically 1–2 Piezo + cells in the cluster (indicated by arrowheads), suggesting that Piezo + cells are generated from ISCs after adulthood. All experiments were independently repeated at least twice with similar results presented in the figures. Data are expressed as mean + s.e.m. P-values are calculated from two-tailed Student t-test with unequal variance. Scale bar: a,c , 50 μm; d,f 20 μm; g , 25 μm.

    Journal: Nature

    Article Title: Mechanical regulation of stem cell differentiation through stretch-activated Piezo channel

    doi: 10.1038/nature25744

    Figure Lengend Snippet: Piezo + /EP cells are ISC-derived EE precursors with reduced mitotic ability. a. Midguts from flies treated with Bleomycin (10 μg/ml Bleo in 5% sucrose) or the γ-secretase inhibitor DAPT (4 mM DAPT in 5% sucrose). Cells that are positive for both Piezo and Pros are indicated by arrowheads. Note that the majority ( > 95%) of Piezo and Pros double positive cells are also positive for esg, suggesting that these cells are “newborn” EEs that still retain esg-GFP signal. b. Percentage of the newborn EEs (Piezo and Pros double positive cells vs. total Pros + EEs) in fly midguts under control, Bleomycin, and DAPT treatments. Cells within 200 μm X 200 μm areas, n=27 (control), n=25 (Bleo), and n=22 (DAPT), were analyzed. c. Midgut with stem cells labeled by esg-GFP (Green), Piezo + cells labeled by Piezo-Gal4 > nlsRFP (Red), and mitotic cells labeled by anti-phospho-Histone H3 (pH3) (Magenta). pH3 + mitotic cell is indicated by arrowhead. d,e. Representative images of midguts from flies feed on either control (5% sucrose) or Bleomycin (5% sucrose plus 10ug/ml Bleomycin) food. Piezo + EP cells are labeled by Piezo-Gal4 > nlsGFP (Green), mitotic cells are labeled by pH3 staining (Red). Mitotic Piezo + cells are indicated by arrowheads. Since all pH3 + cells are Dl + cells (according to Dl-lacZ labeled midgut), we counted all Piezo negative pH3 + cells as pH3 + ISCs. Under both control (5% sucrose) and damage (5% sucrose + 10 μg/ml Bleomycin) conditions, only around 8–10% of the pH3 + cells are Piezo + (~40% of total Dl + cells), suggesting that Piezo + cells are significantly less mitotically active compared to Piezo- Dl + cells. f. If the pH3 + Piezo + cells are also Pros + . Around 50% of these pH3 + Piezo + cells show low levels of Pros staining. Meanwhile, all the pH3 + Pros + cells are positive for Piezo, suggesting that Piezo + EP cells represent more general EE precursor cells compared to EMCs. Mitotic Piezo + cells are indicated by arrowheads. All experiments were independently repeated at least twice with similar results presented in the figures. g. Random GFP + clones were generated using hsFlp; Ubi-(FRT.Stop)GFP/Piezo-Gal4; UAS-nlsRFP . 3–4 days old flies were heat shocked at 37°C for 30 min once to induce clones in ISCs. Then these flies were kept at 25°C for 2 weeks before analysis. Within each GFP + clone, which is derived from ISCs, there are typically 1–2 Piezo + cells in the cluster (indicated by arrowheads), suggesting that Piezo + cells are generated from ISCs after adulthood. All experiments were independently repeated at least twice with similar results presented in the figures. Data are expressed as mean + s.e.m. P-values are calculated from two-tailed Student t-test with unequal variance. Scale bar: a,c , 50 μm; d,f 20 μm; g , 25 μm.

    Article Snippet: The following primary antibodies were used: mouse anti-Prospero (1:50, Developmental Studies Hybridoma Bank, MR1A), rabbit anti-phospho-Histone H3 (Millipore #06–570; 1:1000); mouse anti-HA (Abcam, ab18181), rabbit anti-dpErk1/2 (Cell Signaling #4370; 1:500), mouse anti-Delta (1:50, Developmental Studies Hybridoma Bank, C594.9B), mouse anti-β-galactosidase (1/400, Promega, Z3781), rabbit anti-Tachykinin (1/5000, Veenstra et al. ).

    Techniques: Derivative Assay, Labeling, Staining, Clone Assay, Generated, Two Tailed Test

    Hdac3 overexpression inhibits cyclin-dependent kinase (CDK) inhibitors Cdkn1a (p21 WAF / CIP1 ), Cdkn1b (p27 Kip1 ), Cdkn1c (p57 Kip2 ), Cdkn2c (p18 INC4c ), Cdkn2b (p15 INC4b ), and Cdkn2a ( p16 INC4a ) . A and B , transcript for Cdkn1a , Cdkn1b , Cdkn1c , Cdkn2c , Cdkn2b, and Cdkn2a were detected by qRT-PCR in hearts from P1 ( A ) and 2-month-old ( B ) wild-type and Hdac3 -Tg mice. Three mice in each group were tested, and values are expressed as the -fold change in transcript abundance (±S.D.) when compared with wild-type mice. N.S. , not significant.

    Journal: The Journal of Biological Chemistry

    Article Title: Transgenic Overexpression of Hdac3 in the Heart Produces Increased Postnatal Cardiac Myocyte Proliferation but Does Not Induce Hypertrophy *

    doi: 10.1074/jbc.M803686200

    Figure Lengend Snippet: Hdac3 overexpression inhibits cyclin-dependent kinase (CDK) inhibitors Cdkn1a (p21 WAF / CIP1 ), Cdkn1b (p27 Kip1 ), Cdkn1c (p57 Kip2 ), Cdkn2c (p18 INC4c ), Cdkn2b (p15 INC4b ), and Cdkn2a ( p16 INC4a ) . A and B , transcript for Cdkn1a , Cdkn1b , Cdkn1c , Cdkn2c , Cdkn2b, and Cdkn2a were detected by qRT-PCR in hearts from P1 ( A ) and 2-month-old ( B ) wild-type and Hdac3 -Tg mice. Three mice in each group were tested, and values are expressed as the -fold change in transcript abundance (±S.D.) when compared with wild-type mice. N.S. , not significant.

    Article Snippet: Genotyping was performed by PCR analysis of genomic DNA, and cardiac-specific expression of Hdac3 was revealed by qRT-PCR and Western blot analysis using antibodies to Hdac3 and FLAG (Sigma).

    Techniques: Over Expression, Quantitative RT-PCR, Mouse Assay

    A and B, heart-to-body-weight ratios (A) and heart-weight-to-tibial-length ratios (B) of wild-type and Hdac3-Tg mice at the age of 12 weeks with or without ISO treatment.

    Journal: The Journal of Biological Chemistry

    Article Title: Transgenic Overexpression of Hdac3 in the Heart Produces Increased Postnatal Cardiac Myocyte Proliferation but Does Not Induce Hypertrophy *

    doi: 10.1074/jbc.M803686200

    Figure Lengend Snippet: A and B, heart-to-body-weight ratios (A) and heart-weight-to-tibial-length ratios (B) of wild-type and Hdac3-Tg mice at the age of 12 weeks with or without ISO treatment.

    Article Snippet: Genotyping was performed by PCR analysis of genomic DNA, and cardiac-specific expression of Hdac3 was revealed by qRT-PCR and Western blot analysis using antibodies to Hdac3 and FLAG (Sigma).

    Techniques: Mouse Assay

    Generation and characterization of cardiac myocyte-specific Hdac3-Tg mice. A , schematic of transgenic construct used to generate Hdac3 -Tg mice. α -MHC , α-myosin heavy chain. B , Hdac3 mRNA expression analysis in two different Hdac3 -Tg mice lines. Transcripts for Hdac3 were detected by qRT-PCR in P1 hearts from wild-type ( WT ) and Hdac3 -Tg mice. Three mice in each group were tested, and values are expressed as the -fold change in transcript abundance (± S.D.) when compared with wild-type mice. C , endogenous and transgenic Hdac3 protein were detected with an Hdac3 antibody. All Western blots were performed at least three times with similar results. D , Hdac3 expression in myocardium of P1 wild-type and Hdac3 -Tg mice was quantified by using ImageJ software. E , increased HDAC activity in Hdac3 -Tg mice. Lysates from P1 hearts were assayed for HDAC activity. Data are the average results (± S.D.) from three separate experiments. F , decreased acetylation of histone H4 in Hdac3 -Tg mice. Western blot analysis of acetylated-histone H4 in myocardium from P1 wild-type and Hdac3 -Tg mice using anti-acetyl-histone H4 antibody was performed. ImageJ software was used to quantify -fold change in acetylation. G , Hdac1 and Hdac2 levels are not changed in Hdac3 -Tg mice. Western blot analysis of myocardium lysates from three P1 wild-type and three Hdac3 -Tg mice using anti-Hdac1 and anti-Hdac2 antibody was performed. α-Tubulin is used as a loading control.

    Journal: The Journal of Biological Chemistry

    Article Title: Transgenic Overexpression of Hdac3 in the Heart Produces Increased Postnatal Cardiac Myocyte Proliferation but Does Not Induce Hypertrophy *

    doi: 10.1074/jbc.M803686200

    Figure Lengend Snippet: Generation and characterization of cardiac myocyte-specific Hdac3-Tg mice. A , schematic of transgenic construct used to generate Hdac3 -Tg mice. α -MHC , α-myosin heavy chain. B , Hdac3 mRNA expression analysis in two different Hdac3 -Tg mice lines. Transcripts for Hdac3 were detected by qRT-PCR in P1 hearts from wild-type ( WT ) and Hdac3 -Tg mice. Three mice in each group were tested, and values are expressed as the -fold change in transcript abundance (± S.D.) when compared with wild-type mice. C , endogenous and transgenic Hdac3 protein were detected with an Hdac3 antibody. All Western blots were performed at least three times with similar results. D , Hdac3 expression in myocardium of P1 wild-type and Hdac3 -Tg mice was quantified by using ImageJ software. E , increased HDAC activity in Hdac3 -Tg mice. Lysates from P1 hearts were assayed for HDAC activity. Data are the average results (± S.D.) from three separate experiments. F , decreased acetylation of histone H4 in Hdac3 -Tg mice. Western blot analysis of acetylated-histone H4 in myocardium from P1 wild-type and Hdac3 -Tg mice using anti-acetyl-histone H4 antibody was performed. ImageJ software was used to quantify -fold change in acetylation. G , Hdac1 and Hdac2 levels are not changed in Hdac3 -Tg mice. Western blot analysis of myocardium lysates from three P1 wild-type and three Hdac3 -Tg mice using anti-Hdac1 and anti-Hdac2 antibody was performed. α-Tubulin is used as a loading control.

    Article Snippet: Genotyping was performed by PCR analysis of genomic DNA, and cardiac-specific expression of Hdac3 was revealed by qRT-PCR and Western blot analysis using antibodies to Hdac3 and FLAG (Sigma).

    Techniques: Mouse Assay, Transgenic Assay, Construct, Expressing, Quantitative RT-PCR, Western Blot, Software, Activity Assay

    Cardiac defects in Hdac3-Tg neonates. A , hematoxylin and eosin-stained sections at successive levels of P1 hearts from wild-type ( WT ) and Hdac3 -Tg mice demonstrate a thicker septum, thicker ventricular walls, and diminished ventricular lumens. B , hematoxylin and eosin-stained sections of adult hearts from wild-type and Hdac3 -Tg mice demonstrate normal ventricular septum, walls, and lumens. C , increased proliferation of cardiomyocytes in P1 Hdac3 -Tg mice. Immunohistochemistry for phospho-histone H3 was performed on heart sections from P1 mice. Quantification of phospho-histone H3-positive cells was performed on eight sections from three individual hearts and averaged. D , co-immunofluorescent staining for Ki67 ( red ), MyHC ( green , MF-20 staining), and 4′,6-diamidino-2-phenylindole ( DAPI ) ( blue ) was performed on heart sections from P1 wild-type and Hdac3 -Tg mice. E , wheat germ agglutinin staining shows decreased myocyte size in Hdac3 -Tg when compared with wild-type P1 hearts.

    Journal: The Journal of Biological Chemistry

    Article Title: Transgenic Overexpression of Hdac3 in the Heart Produces Increased Postnatal Cardiac Myocyte Proliferation but Does Not Induce Hypertrophy *

    doi: 10.1074/jbc.M803686200

    Figure Lengend Snippet: Cardiac defects in Hdac3-Tg neonates. A , hematoxylin and eosin-stained sections at successive levels of P1 hearts from wild-type ( WT ) and Hdac3 -Tg mice demonstrate a thicker septum, thicker ventricular walls, and diminished ventricular lumens. B , hematoxylin and eosin-stained sections of adult hearts from wild-type and Hdac3 -Tg mice demonstrate normal ventricular septum, walls, and lumens. C , increased proliferation of cardiomyocytes in P1 Hdac3 -Tg mice. Immunohistochemistry for phospho-histone H3 was performed on heart sections from P1 mice. Quantification of phospho-histone H3-positive cells was performed on eight sections from three individual hearts and averaged. D , co-immunofluorescent staining for Ki67 ( red ), MyHC ( green , MF-20 staining), and 4′,6-diamidino-2-phenylindole ( DAPI ) ( blue ) was performed on heart sections from P1 wild-type and Hdac3 -Tg mice. E , wheat germ agglutinin staining shows decreased myocyte size in Hdac3 -Tg when compared with wild-type P1 hearts.

    Article Snippet: Genotyping was performed by PCR analysis of genomic DNA, and cardiac-specific expression of Hdac3 was revealed by qRT-PCR and Western blot analysis using antibodies to Hdac3 and FLAG (Sigma).

    Techniques: Staining, Mouse Assay, Immunohistochemistry