Journal: Journal of Virology

Article Title: Dysregulation of Cellular VRK1, BAF, and Innate Immune Signaling by the Vaccinia Virus B12 Pseudokinase

doi: 10.1128/jvi.00398-22

Figure Lengend Snippet: VRK1 and B12 coinfluence their solubility properties. (A) Immunoblot analyses of detergent-treated fractions from HAP1 or HAP1-VRK1KO cells transduced with empty-vector (CTRL)- or HA-B12-expressing lentiviruses. Immunoblots detecting Ku70 and histone 2B are shown as fractionation controls. (B) Immunoblot analyses of detergent fractions from CV1, HAP1, or A549 cells uninfected or infected with WT VACV (WR) at an MOI of 5 and harvested at 7 hpi. (C) Immunoblot analyses of fractions from A549 cells uninfected or infected with WT or mutB12 virus (MOI = 5), treated with or without 50 μM AraC, and harvested at 7 hpi. Quantitation of VRK1 and Ku70 abundances was performed for each fraction, with each combined sample abundance (soluble + insoluble) set to 1. Three independent replicates were quantified, (*, P < 0.05). A representative immunoblot is shown.

Article Snippet: Rabbit polyclonal anti-H2B , Santa Cruz Biotechnology , FL-126 , 1:500 , sc-10818.

Techniques: Solubility, Western Blot, Transduction, Plasmid Preparation, Expressing, Fractionation, Infection, Quantitation Assay

Journal: Journal of Virology

Article Title: Dysregulation of Cellular VRK1, BAF, and Innate Immune Signaling by the Vaccinia Virus B12 Pseudokinase

doi: 10.1128/jvi.00398-22

Figure Lengend Snippet: Antibody sources and dilutions

Article Snippet: Rabbit polyclonal anti-H2B , Santa Cruz Biotechnology , FL-126 , 1:500 , sc-10818.

Techniques:

Journal: PLoS ONE

Article Title: Doxorubicin induces large-scale and differential H2A and H2B redistribution in live cells

doi: 10.1371/journal.pone.0231223

Figure Lengend Snippet: (A) Cytometric assay of histone aggregation. Agarose-embedded live Jurkat cells were treated with Dox, then fixed and subsequently permeabilized („Pre-fixed”) or lyzed only. H2A or H2B were detected by indirect immunofluorescence and analyzed by LSC. G 1 cells were gated based on the PI distribution histograms. (B) H2A levels after treatment with different concentrations of Dox, cells lyzed with 1% Triton X-100 in PBS/EDTA after Dox treatment (continuous line), cells pre-fixed and then permeabilized by the same lysing solution (dashed line), cells lyzed with 1% Triton X-100 in sucrose/EDTA (dash-dotted line). The symbols represent measured points, while the lines show the best fit as described in Materials and Methods. LSC settings (photomultiplier voltage gain and offset) were identical during the measurement of all the samples. (C) H2A (continuous line) and H2B (dashed line) levels after treatment with different concentrations of Dox. Fluorescence intensities were normalized to the intensity of untreated samples. In (B) and (C) error bars show SEM values, characterizing population heterogeneity, calculated for the G 1 cell population in a representative experiment. LSC settings were as before. (D) Proposed scheme of Dox-induced aggregation dependent H2A retention. In the permeabilized nuclei, free H2A molecules diffuse out before immunofluorescence labeling, while aggregated and chromatin bound histones remain inside the nuclei. (E) Confocal microscopic images of cells treated with 36 μM Dox and of control cells. After fixation, H2A was detected by immunofluorescence (green) and DNA by PI staining (red). Instrument settings (laser power, photomultiplier voltage, gain, pixel dwell time) and all settings of the image analyses were identical for the compared samples of this panel. Single channel images and an image with amplified DNA signal are shown in .

Article Snippet: The blocking solution was washed out with 500 μl ice cold 1 x PBS/EDTA three times for three minutes and indirect immunofluorescence labeling was performed using mouse monoclonal anti-H2B (ab52484, Abcam, Cambridge, UK; 1 mg/ml), rabbit polyclonal anti-H2B (Sigma-Aldrich; 1 mg/ml) or rabbit polyclonal anti-H2A (ab18255, Abcam, Cambridge, UK; 1 mg/ml).

Techniques: Immunofluorescence, Fluorescence, Labeling, Staining, Amplification

Journal: PLoS ONE

Article Title: Doxorubicin induces large-scale and differential H2A and H2B redistribution in live cells

doi: 10.1371/journal.pone.0231223

Figure Lengend Snippet: (A) Dox concentration dependence of H2B nucleo-cytoplasmic translocation. H2B was detected by immunofluorescence (green), DNA was stained with PI (red) in the fixed Jurkat cells. Representative confocal microscopic images are shown. For single channel images see . (B) Time dependence of Dox-induced H2B nucleo-cytoplasmic translocation. The yellow and blue arrows show depletion of H2B from the central and peripheral nuclear regions, respectively. Control cells incubated in the absence of Dox or labeled only with secondary antibody are also shown. Staining of H2B and of DNA were as in (A). Instrument settings (laser power, photomultiplier voltage, gain, pixel dwell time) and settings of image analyses performed by ImageJ were adjusted so as to detect also cytoplasmic H2B in the control cells and were identical for each compared sample of a particular experiment, both in the case of panel A and B. For single channel images see . (C), (D) Quantification of fluorescence microscopic images of panel A and B, respectively. The total cytoplasmic H2B immunofluorescence was determined as described in Materials and Methods and normalized to the total cellular immunofluorescence. Error bars represent SEM.

Article Snippet: The blocking solution was washed out with 500 μl ice cold 1 x PBS/EDTA three times for three minutes and indirect immunofluorescence labeling was performed using mouse monoclonal anti-H2B (ab52484, Abcam, Cambridge, UK; 1 mg/ml), rabbit polyclonal anti-H2B (Sigma-Aldrich; 1 mg/ml) or rabbit polyclonal anti-H2A (ab18255, Abcam, Cambridge, UK; 1 mg/ml).

Techniques: Concentration Assay, Translocation Assay, Immunofluorescence, Staining, Incubation, Labeling, Fluorescence

Journal: PLoS ONE

Article Title: Doxorubicin induces large-scale and differential H2A and H2B redistribution in live cells

doi: 10.1371/journal.pone.0231223

Figure Lengend Snippet: ( A) The cells co-treated with the drug(s) indicated in the Figure and with 36 μM Dox for 2 hrs were fixed and stained as in . H2B was labeled with a monoclonal anti-H2B antibody, except for the samples marked with an asterisk (*) that were labeled with a polyclonal anti-H2B antibody (see ). # marks the sample treated with both 50 μM PYR-41 and 100 μM 2-D08 (upper image), or co-treated with the two inhibitors and 36 μM Dox (lower image). DNA was stained with PI. H2B immunofluorescence: green; PI: red. Representative confocal microscopic images are shown. Instrument settings and settings of image analyses performed by ImageJ were identical for each compared sample of a particular experiment. Single channel images are shown in . (B) Quantification of fluorescence microscopic images of panel A. The total cytoplasmic H2B immunofluorescence was determined as described in Materials and Methods and normalized to the total cellular immunofluorescence. Error bars represent SEM. * indicates significant difference based on two-tailed Student’s t-test (p<0.001).

Article Snippet: The blocking solution was washed out with 500 μl ice cold 1 x PBS/EDTA three times for three minutes and indirect immunofluorescence labeling was performed using mouse monoclonal anti-H2B (ab52484, Abcam, Cambridge, UK; 1 mg/ml), rabbit polyclonal anti-H2B (Sigma-Aldrich; 1 mg/ml) or rabbit polyclonal anti-H2A (ab18255, Abcam, Cambridge, UK; 1 mg/ml).

Techniques: Staining, Labeling, Immunofluorescence, Fluorescence, Two Tailed Test

Journal: PLoS ONE

Article Title: Doxorubicin induces large-scale and differential H2A and H2B redistribution in live cells

doi: 10.1371/journal.pone.0231223

Figure Lengend Snippet: (A) Upper row: Dox concentration dependence of H2B nucleo-cytoplasmic translocation. Lower row: H2A immunofluorescence in the same cells that are shown in the upper row. The cells were treated with Dox alone or with Dox and 50 μM PYR-41 together for 2 hrs. DNA was stained with PI. The colors are as in the previous Figures. Representative confocal microscopic images are shown. Instrument settings and settings of image analyses performed by ImageJ were identical for each compared sample of a particular experiment. Single channel images are included in . (B) Quantification of fluorescence microscopic images of panel A. The total cytoplasmic H2B immunofluorescence was determined as described in Materials and Methods and normalized to the total cellular immunofluorescence. Error bars represent SEM.* indicates significant difference based on the two-tailed Student’s t-test (p<0.001).

Article Snippet: The blocking solution was washed out with 500 μl ice cold 1 x PBS/EDTA three times for three minutes and indirect immunofluorescence labeling was performed using mouse monoclonal anti-H2B (ab52484, Abcam, Cambridge, UK; 1 mg/ml), rabbit polyclonal anti-H2B (Sigma-Aldrich; 1 mg/ml) or rabbit polyclonal anti-H2A (ab18255, Abcam, Cambridge, UK; 1 mg/ml).

Techniques: Concentration Assay, Translocation Assay, Immunofluorescence, Staining, Fluorescence, Two Tailed Test

Journal: PLoS ONE

Article Title: Doxorubicin induces large-scale and differential H2A and H2B redistribution in live cells

doi: 10.1371/journal.pone.0231223

Figure Lengend Snippet: (A) Cells treated with the drug(s) indicated in the Figure for 2 hrs were fixed and labeled with a monoclonal anti-H2B antibody (green) and the DNA was stained by PI (red). Representative confocal microscopic images are shown. Instrument settings and settings of image analyses performed by ImageJ were identical for each compared sample of a particular experiment. More images are shown in . (B) Quantification of fluorescence microscopic images of panel A. The total nuclear H2B immunofluorescence was determined as described in Materials and Methods and normalized to the total cellular immunofluorescence. Error bars represent SEM.* indicates significant difference based on the two-tailed Student’s t-test (p<0.001).

Article Snippet: The blocking solution was washed out with 500 μl ice cold 1 x PBS/EDTA three times for three minutes and indirect immunofluorescence labeling was performed using mouse monoclonal anti-H2B (ab52484, Abcam, Cambridge, UK; 1 mg/ml), rabbit polyclonal anti-H2B (Sigma-Aldrich; 1 mg/ml) or rabbit polyclonal anti-H2A (ab18255, Abcam, Cambridge, UK; 1 mg/ml).

Techniques: Labeling, Staining, Fluorescence, Immunofluorescence, Two Tailed Test

Journal: PLoS ONE

Article Title: Doxorubicin induces large-scale and differential H2A and H2B redistribution in live cells

doi: 10.1371/journal.pone.0231223

Figure Lengend Snippet: Histones detected by MS in the supernatant of Dox-treated, Dox + PYR-41-treated and untreated samples.

Article Snippet: The blocking solution was washed out with 500 μl ice cold 1 x PBS/EDTA three times for three minutes and indirect immunofluorescence labeling was performed using mouse monoclonal anti-H2B (ab52484, Abcam, Cambridge, UK; 1 mg/ml), rabbit polyclonal anti-H2B (Sigma-Aldrich; 1 mg/ml) or rabbit polyclonal anti-H2A (ab18255, Abcam, Cambridge, UK; 1 mg/ml).

Techniques:

Journal: PLoS ONE

Article Title: Loss of the Histone Pre-mRNA Processing Factor Stem-Loop Binding Protein in Drosophila Causes Genomic Instability and Impaired Cellular Proliferation

doi: 10.1371/journal.pone.0008168

Figure Lengend Snippet: Protein lysates from wt, Slbp 15 , Slbp 10 , and H2aV 810 mutants were obtained from whole 3 rd instar larvae and probed with antibodies to A) H2b and H3, and B) H3K4-me2 and H3K9-me2. β-tubulin was used as a loading control for both panels.

Article Snippet: Polyclonal rabbit C-terminal H3 (1∶3000) (Abcam #1791), polyclonal rabbit H2b (Abcam #1790) (1∶3000), monoclonal mouse H3K9-me2 (Abcam #1220) (1∶750), and polyclonal rabbit H3K4-me2 (Abcam #7766) (1∶3000), and monoclonal mouse Β-tubulin (1∶1000) antibodies were obtained commercially.

Techniques: