Journal: Journal of Nanomaterials

Article Title: Preparation and Characterization of Emamectin Benzoate Solid Nanodispersion

doi: 10.1155/2017/6560780

Figure Lengend Snippet: Figure 2: (a) SEM image of emamectin benzoate nanoparticles and (b) particle size distribution of emamectin benzoate SNDs measured by DLS.

Article Snippet: morphology of emamectin benzoate solid nanodispersion was characterized by a JSM-7401F scanning electron microscopy (SEM) (JEOL Ltd., Japan) with an accelerating voltage of 3 kV.

Techniques:

Journal: Journal of Virology

Article Title: Hepatitis C Virus Nonstructural Protein 5A Modulates the Toll-Like Receptor-MyD88-Dependent Signaling Pathway in Macrophage Cell Lines

doi: 10.1128/jvi.00649-07

Figure Lengend Snippet: FIG. 1. Establishment of stable macrophage cell lines expressing HCV proteins. (A) Cell lysates were prepared from macrophage cell lines expressing each of the HCV proteins (4 106 cells) and immunoblotted with antibodies against HCV proteins or -actin. (B) Total RNA was extracted from macrophage cell lines expressing NS5A (gray bars) or control (white bars), and the expression of mRNA of TLRs was determined by real-time PCR. (C) The subcellular localization of NS5A was examined by confocal microscopy. Cells were fixed with 4% paraformaldehyde– PBS, permeabilized with 0.5% Triton X-100, and stained with specific antibodies. Cells expressing NS5A or control cells were extracted into cytosol (C), membrane-organelle (M), and nuclear (N) fractions. Each fraction was concentrated and subjected to immunoblotting with specific antibodies. PA28, calregulin, and histone H1 were used as markers for cytosol, membrane-organelle, and nuclear fractions, respectively.

Article Snippet: PA28 (Biomol International, Plymouth Meeting, PA), calregulin, and histone H1 (Santa Cruz Biotechnology) were used as cytoplasmic, membrane, and nuclear markers, respectively.

Techniques: Expressing, Control, Real-time Polymerase Chain Reaction, Confocal Microscopy, Staining, Membrane, Western Blot

Journal: Nature Communications

Article Title: Functional interplay between TFIIH and KAT2A regulates higher-order chromatin structure and class II gene expression

doi: 10.1038/s41467-019-09270-2

Figure Lengend Snippet: A loss of XPB NTD integrity induces large-scale chromatine decondensation. a Schematic representation of the lacO/LacR tethering system used in U2OS17 cells. See the Materials and methods section for a full description of the cell line. b Schematic representation of wild-type and mutant XPB-LacR-GFP constructs. For clarity, the sizes of the GFP (238 aa) and LacR (367 aa) are omitted. c Proteins from whole-cell extracts (15 μg) of U2OS17 cells transiently transfected with wild-type or mutant XPB-LacR-GFP constructs were resolved by SDS-PAGE and immunoblotted using either polyclonal rabbit anti-GFP (upper panel), polyclonal rabbit anti-XPB (middle panel) or monoclonal mouse anti-Actin antibodies (lower panel). Source data are provided as a Source Data file. d U2OS17 cells were transiently transfected with 1 μg of expression vectors for the following proteins: LacR-GFP, XPB WT -LacR-GFP, XPB 320–782 -LacR-GFP, XPB F99S -LacR-GFP, XPB T119P -LacR-GFP, XPB 1–550 -LacR-GFP. In parallel, U2OS17 cell line was transiently transfected with 5 μg of expression vector for LacR-GFP. GFP was observed by fluorescence microscopy 24 h post transfection. Lower panels are magnifications of the white rectangles in the upper panels. e 3D reconstruction of U2OS17 cellular nuclei transiently transfected either with XPB WT -LacR-GFP or with XPB F99S -LacR-GFP using Imaris Software (Bitplane). f Whisker box plot shows quantifications of the relative array volumes (volume of the array/volume of the nucleus; 40–150 cells for each condition). Significant p -values are indicated (*** ≤ 0.001) and were obtained using a Kruskal Wallis test. Source data are provided as a Source Data file

Article Snippet: Lower panels are magnifications of the white rectangles in the upper panels. e 3D reconstruction of U2OS17 cellular nuclei transiently transfected either with XPB WT -LacR-GFP or with XPB F99S -LacR-GFP using Imaris Software (Bitplane). f Whisker box plot shows quantifications of the relative array volumes (volume of the array/volume of the nucleus; 40–150 cells for each condition).

Techniques: Mutagenesis, Construct, Transfection, SDS Page, Expressing, Plasmid Preparation, Fluorescence, Microscopy, Software, Whisker Assay

Journal: Nature Communications

Article Title: Functional interplay between TFIIH and KAT2A regulates higher-order chromatin structure and class II gene expression

doi: 10.1038/s41467-019-09270-2

Figure Lengend Snippet: XP-B/CS F99S patient-derived cells have a global increase in the H3K9ac histone mark. a Extracts from either XP-B/CS F99S or XP-B/CS F99S + XPB WT cells were resolved by SDS-PAGE and immunoblotted with a monoclonal mouse anti-XPB antibody. Source data are provided as a Source Data file. (6-4)PP removal measurements were carried out in XP-B/CS F99S , stably transfected XP-B/CS F99S + XPB WT and wild-type MRC5 cells harvested at different time points after UV irradiation at 30 J/m 2 as indicated. Cells were labeled with a monoclonal mouse anti-(6-4)PP antibody and signals were measured using a INCell 1000 analyzer (GE Healthcare). The graph represents the percentage of lesions remaining in the genome at a given time (error bars represent SD from three independent experiments). For each time point, about 20000–40000 cells were analyzed. b Histones were extracted from either XP-B/CS F99S , XP-B/CS F99S + XPB WT or wild-type MRC5 fibroblasts, resolved by SDS-PAGE and immunoblotted with either polyclonal rabbit anti-histone H3, polyclonal rabbit anti-histone H3ac, monoclonal mouse anti-histone H3K9ac or polyclonal rabbit anti-histone H3K9me2. Source data are provided as a Source Data file. c XP-B/CS F99S or XP-B/CS F99S + XPB WT cells were treated 3 h with DMSO or SP (50 μM) and histones were extracted, resolved by SDS-PAGE and immunoblotted either with a polyclonal rabbit anti-histone H3 or with monoclonal mouse anti-histone H3K9ac antibodies. In parallel, cell extracts were resolved by SDS-PAGE and western blotted with either a monoclonal mouse anti-XPB or polyclonal rabbit anti-tubulin antibodies. Source data are provided as a Source Data file

Article Snippet: Lower panels are magnifications of the white rectangles in the upper panels. e 3D reconstruction of U2OS17 cellular nuclei transiently transfected either with XPB WT -LacR-GFP or with XPB F99S -LacR-GFP using Imaris Software (Bitplane). f Whisker box plot shows quantifications of the relative array volumes (volume of the array/volume of the nucleus; 40–150 cells for each condition).

Techniques: Derivative Assay, SDS Page, Stable Transfection, Transfection, Irradiation, Labeling, Western Blot

Journal: Nature Communications

Article Title: Functional interplay between TFIIH and KAT2A regulates higher-order chromatin structure and class II gene expression

doi: 10.1038/s41467-019-09270-2

Figure Lengend Snippet: XPB recruits KAT2A-containing HAT complexes to chromatin. a TFIIH-XPB WT was immunoprecipitated from nuclear extracts of stably transfected cells using anti-GFP antibodies (GFP-Trap) and washed with 200 mM salt (Lanes 2 and 4). Control IPs were performed with anti-GFP on an XP-B/CS F99S cell line stably expressing GFP alone (Lanes 1 and 3). Proteins on the resin were resolved by SDS-PAGE and immunoblotted using polyclonal rabbit anti-GFP, monoclonal mouse anti-p62, polyclonal rabbit anti-CDK7, polyclonal rabbit anti-KAT2A (SCBT) (Supplementary Figure ), polyclonal rabbit anti-KAT2B, polyclonal rabbit anti-WDR5, or polyclonal rabbit anti-SUPT7L antibodies. Source data are provided as a Source Data file. b The U2OS17 cell line was transiently transfected with 1 μg of expression vectors for the following proteins: LacR-GFP, XPB WT -LacR-GFP, XPB F99S -LacR-GFP, or XPB 320–782 -LacR-GFP together with 1 μg of expression vector for Flag-KAT2A. Colocalization of KAT2A with GFP was detected by immunofluorescence staining using a polyclonal rabbit anti-flag antibody. The values on the graph represent the percentage of colocalization of KAT2A with GFP on the array (error bars represent SD from three independent quantifications). c Purified wild-type recombinant core TFIIH (cIIH-XPB WT ) was incubated with purified Flag-KAT2A (400 ng) and pull-down assays were performed using either unspecific anti-IgG or polyclonal rabbit anti-KAT2A (SCBT) antibody (Supplementary Figure ). After washing, proteins on the resin were resolved by SDS-PAGE and immunoblotted using mouse monoclonal anti-XPB or anti-p52 antibodies (two subunits of TFIIH) or monoclonal mouse anti-KAT2A (IGBMC) antibody. Source data are provided as a Source Data file. d Bacterially expressed recombinant KAT2A (rKAT2A) (400 ng) was tested for its ability to interact with either rXPB WT (lanes 1–3) or rXPB F99S (lanes 4–6) (500 ng) in a pulled-down assay (IP-XPB). After washing, proteins on the resin were resolved by SDS-PAGE and immunoblotted using mouse monoclonal anti-XPB and mouse monoclonal anti-KAT2A antibodies (IGBMC). Lanes 1 and 4, immunoprecipitations with an irrelevant antibody (IP-IgG). Source data are provided as a Source Data file

Article Snippet: Lower panels are magnifications of the white rectangles in the upper panels. e 3D reconstruction of U2OS17 cellular nuclei transiently transfected either with XPB WT -LacR-GFP or with XPB F99S -LacR-GFP using Imaris Software (Bitplane). f Whisker box plot shows quantifications of the relative array volumes (volume of the array/volume of the nucleus; 40–150 cells for each condition).

Techniques: Immunoprecipitation, Stable Transfection, Transfection, Control, Expressing, SDS Page, Plasmid Preparation, Immunofluorescence, Staining, Purification, Recombinant, Incubation

Journal: Nature Communications

Article Title: Functional interplay between TFIIH and KAT2A regulates higher-order chromatin structure and class II gene expression

doi: 10.1038/s41467-019-09270-2

Figure Lengend Snippet: Loss of XPB NTD integrity induces an increase in KAT2A HAT activity. a rKAT2A and the recombinant HAT-ATAC module (rHAT-ATAC) containing KAT2A, ADA3, ADA2a, and SGF29 were resolved by SDS-PAGE followed by Coomassie staining. Core TFIIH containing p62, p52, p44, p34 and either XPB WT (cIIH-XPB WT ) or XPB F99S (cIIH-XPB F99S ) were resolved by SDS-PAGE followed by Coomassie staining. Source data are provided as a Source Data file. b One hundred nanograms of core TFIIH containing either XPB WT (cIIH-XPB WT ) or XPB F99S (cIIH-XPB F99S ) were incubated with 50 ng rKAT2A together with histone H3.3 and cold acetyl-CoA. Following resolution by SDS-PAGE, proteins were immunoblotted using polyclonal rabbit anti-H3, monoclonal mouse anti-H3K9Ac, mouse monoclonal anti-XPB, and polyclonal rabbit anti-KAT2A (SCBT) antibodies. Quantification of H3K9Ac was performed using ImageJ software and normalized with H3. Source data are provided as a Source Data file. c Twenty nanograms of rXPB WT or rXPB F99S were incubated with 50 ng of rKAT2A together with histone H3.3 and cold acetyl-CoA. Following incubation, reactions were treated as described in panel ( b ). Source data are provided as a Source Data file. d Twenty nanograms of rXPB WT or rXPB F99S were incubated with 200 ng of the rHAT-ATAC module together with histone H3.3 and cold Acetyl-CoA. Following incubation, reactions were treated as described in panel ( b ). Source data are provided as a Source Data file

Article Snippet: Lower panels are magnifications of the white rectangles in the upper panels. e 3D reconstruction of U2OS17 cellular nuclei transiently transfected either with XPB WT -LacR-GFP or with XPB F99S -LacR-GFP using Imaris Software (Bitplane). f Whisker box plot shows quantifications of the relative array volumes (volume of the array/volume of the nucleus; 40–150 cells for each condition).

Techniques: Activity Assay, Recombinant, SDS Page, Staining, Incubation, Software

Journal: Nature Communications

Article Title: Functional interplay between TFIIH and KAT2A regulates higher-order chromatin structure and class II gene expression

doi: 10.1038/s41467-019-09270-2

Figure Lengend Snippet: Higher-order chromatin decondensation induced by XPB mutants is due to KAT2A HAT activity. a U2OS17 cells were transfected either with siCTL or with siKAT2A for 72 h and cell extracts were resolved by SDS-PAGE and immunoblotted using polyclonal rabbit anti-KAT2A or polyclonal rabbit anti-tubulin antibodies. siCTL or siKAT2A transfected cells were re-transfected with either LacR-GFP, XPB WT -LacR-GFP, XPB F99S -LacR-GFP or XPB 320–782 -LacR-GFP for 24 h and the relative array volumes (Vol) were quantified as described above. Significant p -value are indicated (*** ≤ 0.001) and were obtained using a Kruskal Wallis test. Source data are provided as a Source Data file. b XP-B/CS F99S and XP-B/CS F99S + XPB WT cells were treated or not with MB-3 (200 μM) for 15 h and were subsequently labeled with polyclonal rabbit anti-tubulin and stained with DAPI. Cells were then reconstructed in 3D using Imaris Software (Bitplane) and the volumes of the cells and their nuclei were measured. The whisker box plot shows the volume of the nucleus/volume (Vol) of the cell (at least 30 cells for each condition, error bars represent SD from three independent experiments). The significant p -value is indicated (*** ≤ 0.001) and were obtained using a Kruskal Wallis test. Source data are provided as a Source Data file

Article Snippet: Lower panels are magnifications of the white rectangles in the upper panels. e 3D reconstruction of U2OS17 cellular nuclei transiently transfected either with XPB WT -LacR-GFP or with XPB F99S -LacR-GFP using Imaris Software (Bitplane). f Whisker box plot shows quantifications of the relative array volumes (volume of the array/volume of the nucleus; 40–150 cells for each condition).

Techniques: Activity Assay, Transfection, SDS Page, Labeling, Staining, Software, Whisker Assay

Journal: Nature Communications

Article Title: Functional interplay between TFIIH and KAT2A regulates higher-order chromatin structure and class II gene expression

doi: 10.1038/s41467-019-09270-2

Figure Lengend Snippet: KAT2A HAT activity induces inappropriate gene activation in XP-B/CS F99S cells. a RNA-seq analysis scatter plots comparing XP-B/CS F99S vs XP-B/CS F99S + XPB WT transcription profiles. Points show significantly over- (bottom) or under-(top) represented mRNA in XP-B/CS F99S cells compared to XP-B/CS F99S + XPB WT . All data were evaluated with the DESeq2 R package. For a given gene, its value is the normalized gene expression value relative to the mean of all samples belonging to the same condition. b A Comparative analysis of RNA-seq data from XP-B/CS F99S and XP-B/CS F99S + XPB WT . The pie chart indicates the number of genes that are up- and downregulated in the XP-B/CS F99S vs XP-B/CS F99S + XPB WT . c Pre-mRNA levels (error bars represent SD from three independent experiments) of LRRC42, NETO1, RNF130 , RARβ2 , and CYP26 were analyzed in XP-B/CS F99S or XP-B/CS F99S + XPB WT cells treated with either DMSO or DRB, as indicated. Data represent the relative expression levels of the pre-mRNA vs. GAPDH mRNA. Source data are provided as a Source Data file. d Pre-mRNA levels (error bars represent SD from three independent experiments) of LRRC42, NETO1, RNF130 , RARβ2 , and CYP26 were analyzed in XP-B/CS F99S or XP-B/CS F99S + XPB WT cells treated either with DMSO or with MB-3 (200 μM) as indicated. Data represent the relative expression levels of the pre-mRNA vs. GAPDH mRNA. Source data are provided as a Source Data file. e Pre-mRNA levels (error bars represent SD from three independent experiments) of LRRC42, NETO1, RNF130 , RARβ2 , and CYP26 were analyzed in XP-B/CS F99S or XP-B/CS F99S + XPB WT cells treated with either siCTL or siKAT2A, as indicated. Data represent the relative expression levels of the pre-mRNA vs. GAPDH mRNA. Source data are provided as a Source Data file. f Pre-mRNA levels (error bars represent SD from three independent experiments) of CYP26 and LRRC42 were analyzed in XP-B/CS F99S and XP-B/CS F99S + XPB WT cells as well as on stable clone 14 and clone 5 that were selected after transfection of cDNA coding for XPB T119P . Clone 14 does not express the transgene while clone 5 expresses only the transgene. Data represent the relative expression levels of the pre-mRNA vs. GAPDH mRNA. Source data are provided as a Source Data file

Article Snippet: Lower panels are magnifications of the white rectangles in the upper panels. e 3D reconstruction of U2OS17 cellular nuclei transiently transfected either with XPB WT -LacR-GFP or with XPB F99S -LacR-GFP using Imaris Software (Bitplane). f Whisker box plot shows quantifications of the relative array volumes (volume of the array/volume of the nucleus; 40–150 cells for each condition).

Techniques: Activity Assay, Activation Assay, RNA Sequencing, Gene Expression, Expressing, Stable Transfection, Transfection

Journal: Nature Communications

Article Title: Functional interplay between TFIIH and KAT2A regulates higher-order chromatin structure and class II gene expression

doi: 10.1038/s41467-019-09270-2

Figure Lengend Snippet: Chromatin modification and RNA Pol II TIC formation at the promoters of overexpressed genes in XP-B/CS cells. a , b Schematic representations of the RARβ2 ( a ) and RNF130 ( b ) gene regions, including the upstream (Us) and promoter (Pr 1 and 2, respectively) regions that were amplified (see in facing half-arrowheads). c – j ChIPs monitoring occupancy by KAT2A (using a polyclonal rabbit anti-KAT2A antibody (EpiGentek) Supplementary Figure ) ( c , d ), H3K9ac/H3 (using monoclonal mouse anti-H3K9Ac and polyclonal rabbit anti-H3 antibodies) ( e , f ), TFIIB (using a polyclonal rabbit anti-TFIIB antibody) ( g , h ) and Pol II pS5 (using a monoclonal rat anti-Pol II pS5 antibody) ( i , j ) of either the Us or the Pr regions of RARβ2 ( c , e , g , i ) or the Pr region of RNF130 ( d , f , h , j ) in XP-B/CS F99S and XP-B/CS F99S + XPB WT cells treated either with DMSO or with MB-3 (200 μM). k – n ChIPs monitoring occupancy by the TFIIH core subunits p62 (using polyclonal rabbit anti-p62 antibody) ( k , l ) and XPB F99S (using polyclonal rabbit anti-XPB antibody) ( m , n ) of the Us or Pr regions of RARβ2 ( k – m ) or of the Pr region of RNF130 ( l – n ) in XP-B/CS F99S treated with either DMSO or MB-3

Article Snippet: Lower panels are magnifications of the white rectangles in the upper panels. e 3D reconstruction of U2OS17 cellular nuclei transiently transfected either with XPB WT -LacR-GFP or with XPB F99S -LacR-GFP using Imaris Software (Bitplane). f Whisker box plot shows quantifications of the relative array volumes (volume of the array/volume of the nucleus; 40–150 cells for each condition).

Techniques: Modification, Amplification