Journal: Nucleus

Article Title: Gene-rich chromosomal regions are preferentially localized in the lamin B deficient nuclear blebs of atypical progeria cells

doi: 10.1080/19491034.2015.1004256

Figure Lengend Snippet: Bleb-associated genetic regions are actively transcribed. ( A ) Primary p.S143F fibroblasts (p33) were stained for either RNA polymerase II (first row) or active RNA polymerase II (Pol IIo, second row), LA and LB1. Hoechst was used to visualize DNA. Single mid-plane confocal sections from the whole cell are shown. RNA polymerase II and active RNA polymerase II (Pol IIo) are enriched in the blebs (arrows). Scale bars 5 μm. ( B ) Transcriptional activity in p.S143F fibroblasts (p30) was visualized by BrUTP incorporation into nascent RNA by scratch labeling. DAPI was used to visualize DNA. A single mid-plane confocal section is shown. Note the intense BrUTP staining in the bleb compartment (arrow). Scale bar 5 μm. ( C ) p.S143F fibroblasts (p36) were subjected to RNA FISH with a probe for a highly transcribed region on chromosome 19. DAPI was used to visualize DNA. Single confocal sections from mid-plane and nuclear surface are shown. FISH signals are detectable in the bleb region (arrow). Scale bar 5 μm. ( D ) p.S143F fibroblasts (p21) were stained for LA/C and histone H3 trimethylated at lysine 4 or lysine 36. Hoechst was used to visualize DNA. Single mid-plane confocal sections are shown. Both histone marks are detectable in the bleb region (arrows). Scale bars 5 μm.

Article Snippet: Primary antibodies used were mouse monoclonal anti-LA/C (1:300; JoL2, Chemicon, cat.# MAB3211), rabbit polyclonal anti-LA (1:1000) , rabbit polyclonal anti-LB1 (1:1000) , mouse monoclonal anti-LB2 (1:100; LN43, Abcam, cat.# ab8983), rabbit polyclonal anti-H3K9me3 (1:1000, Abcam, cat.# ab8898) and rabbit polyclonal anti-H3K27me3 (1:1000, Abcam, cat.# ab108245), rabbit polyclonal anti-H3K4me2 (1:1000, Abcam, cat.# ab7766), rabbit polyclonal anti-H3K36me3 (1:1000, Abcam, cat.# ab9050, rabbit polyclonal anti-AcH3 (1:1000, Upstate/Merck Millipore, cat.# 06–599), mouse monoclonal anti-Pol II (1:1000, 4H8, Abcam, cat.# ab5408), mouse monoclonal anti-Pol II phosphorylated at Ser2 (H5, 1:100, Abcam, cat.# ab24758), mouse monoclonal anti-FLAG (1:400, clone M2, Sigma, cat.# F1804), rabbit polyclonal anti-SUN1 and anti-SUN2 (1:40 and 1:20; kindly provided by Didier Hodzic) , goat polyclonal anti-LB1 (1:200 Santa Cruz cat.# sc-6217), mouse anti-BrUTP (1:100, Caltag Laboratories, clone Br-3), rabbit polyclonal anti-p53 (1:100, Imgenex, cat.# IMG-533) and rabbit polyclonal anti-SKIP (NCOA62) (1:100, Abcam, cat.# ab153887).

Techniques: Staining, Activity Assay, Labeling, Fluorescence In Situ Hybridization

Journal: Genes & Development

Article Title: The Drosophila P68 RNA helicase regulates transcriptional deactivation by promoting RNA release from chromatin

doi: 10.1101/gad.1396306

Figure Lengend Snippet: p68 mutation blocks gene shutoff during heat shock. ( A–D ) Control ( A,C ) and CB02119/Lip H ( B,D ) larvae were heat-shocked at 37°C for 20 min and immediately processed. The cells were stained using anti-H5 antibodies (green) and anti-HSF antibodies (red) (cf. C and D ). Examining the anti-H5 staining alone reveals that active Pol II is limited to major heat-shock puffs in control cells ( C ), while in p68 mutant cells, it is present at heat-shock loci, other discrete sites, and generally along chromosome arms ( D ) ( hsp70 gene loci at 87A and 87C are indicated). ( E–F ) Control ( E,G ) and CB02119/Lip H ( F,H ) larvae were heat-shocked at 37°C for 10 min, pulse-labeled with Br-UTP, and heat-shocked for an additional 20 min. ( E,G ) Control samples exhibit Br-UTP labeling only at heat-shock response puffs. ( F,H ) p68 mutant cells show high levels of Br-UTP incorporation at many chromosomal sites including the nucleolus, demonstrating that a broad number of genes continue to transcribe RNA during heat shock in the absence of p68.

Article Snippet: A final concentration of 2 mM Br-UTP (Sigma) was used to pulse-label nascent RNA in salivary glands.

Techniques: Mutagenesis, Staining, Labeling

Journal: Genes & Development

Article Title: The Drosophila P68 RNA helicase regulates transcriptional deactivation by promoting RNA release from chromatin

doi: 10.1101/gad.1396306

Figure Lengend Snippet: p68 mutants display RNA export defects. ( A,B ) Control ( A ) and CB02119/Lip F ( B ) transheterozygotes labeled for hsp70 RNA immediately after a 20-min heat shock. The edge of representative nuclei is outlined in white (boxes). p68 mutant salivary glands have less cytoplasmic hsp70 RNA and qualitatively higher levels of hsp70 RNA at transcription sites. ( C,D ) Control ( C ) and CB02119/Lip F ( D ) salivary glands labeled with Br-UTP. Control cells exhibit cytoplasmic staining and punctuate nuclear Br-UTP staining, whereas p68 mutant salivary glands display robust Br-UTP labeling on polytene chromosomal bands. Little signal was observed in the cytoplasm of mutant cells. ( E–H ) Control ( E,G ) and CB02119/Lip F ( F,H ) salivary gland cells stained with anti-SBR (NXF1) antibodies ( E,F ) or anti-ALY (REF1) antibodies ( G,H ). SBR and ALY are found along the nuclear periphery and in the nucleoplasm of control cells. In p68 mutant cells, their distribution is changed, and both export factors accumulate in the nuclear interior.

Article Snippet: A final concentration of 2 mM Br-UTP (Sigma) was used to pulse-label nascent RNA in salivary glands.

Techniques: Labeling, Mutagenesis, Staining

Journal: Neuro-Oncology

Article Title: uPAR and cathepsin B inhibition enhanced radiation-induced apoptosis in gliomainitiating cells

doi: 10.1093/neuonc/nos088

Figure Lengend Snippet: Effect of pCU on run-on transcription. (A–B) Simultaneous visualization of transcription (green) and γH2AX (red) in pCU-treated U87 and 4910 non-GICs and GICs with and without radiation. Cells were transfected with pCU alone or pCU plus radiation and incubated with BrUTP at the final concentration of 5 mM. Cells were fixed and stained with anti-BrdU antibody and anti-phospho H2AX antibodies for 1 h and counterstained with species-specific Alexa Fluor-conjugated secondary antibodies for 1 h. Before mounting, cells were treated with DAPI and analyzed under a confocal microscope (Olympus BX61 Fluoview). Overlay of images was done using SPOT Advanced software (Windows version 4.0.8). (C–D) Expression of survivin mRNA. Total RNA was extracted from both non-GICs and GICs, and mRNA expression of survivin was determined by RT-PCR. GAPDH was used as a loading control. (E–F) Expression of survivin protein. Cell lysates from pCU-treated U87 and 4910 non-GICs and GICs with and without radiation were analyzed for expression of survivin by Western blotting. (G–H) Expression of survivin mRNA after transfection with full-length H2AX (FLH). Total RNA was extracted from both non-GICs and GICs, and mRNA expression of H2AX and survivin was determined by RT-PCR. (I–J) Lysates from FLH-treated cells with or without radiation were analyzed for γH2AX, H2AX and survivin proteins by Western blotting.

Article Snippet: Here, GICs and non-GICs were plated onto 4-well chamber slides and treated with pCU and radiation as described previously, and 150 µL of medium containing BrUTP (SC: 70443, Santa Cruz Biotechnology) at a final concentration of 5 mM were added to each well.

Techniques: Transfection, Incubation, Concentration Assay, Staining, Microscopy, Software, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Journal of Virology

Article Title: Functional Characterization of Negri Bodies (NBs) in Rabies Virus-Infected Cells: Evidence that NBs Are Sites of Viral Transcription and Replication ▿

doi: 10.1128/JVI.00554-09

Figure Lengend Snippet: Newly synthesized viral RNAs are present inside the viral IBs. (A) BSR cells were infected at an MOI of 3 for 16 h p.i., transfected with BrUTP for 20 min in the presence (+ActD) or absence (-ActD) of ActD. P protein (as a marker of the Negri bodies) was detected by using the rabbit polyclonal anti-P antibody, followed by incubation with Alexa 568-goat anti-rabbit IgG (P). Newly synthesized RNAs were detected by using mouse MAb anti-BrdU antibody (BrUTP) and goat anti-mouse Alexa 488. Colocalization of the viral RNA (vRNA) with the IBs is shown in the merged panel in the presence of the drug (+ActD). In cells untreated with ActD, cellular mRNA is produced in vast excess over vRNA. Thus, for these images, the PMT gain of the confocal laser scanning microscope is lower, so the signal for vRNA is lower than for that shown in the presence of the drug, and specific colocalization is not readily observed. The scale bars correspond to 12 μm. (B) BrUTP signals are specific to RNA produced by rabies virus. BSR cells were infected with the VTF7-3 recombinant virus and cotransfected with plasmids expressing N and P proteins. Cells were then transfected with BrUTP for 20 min in the presence (+ActD) or absence (-ActD) of ActD. P protein and RNA were detected as in panel A. Note that in the absence of the drug, no BrUTP signal is detectable in the IBs formed by N and P proteins, but some RNA signals are detected in the cytoplasmic inclusions, most probably due to vaccinia virus infection (-ActD). In the presence of the drug, no RNA labeling is observed (+ActD).

Article Snippet: Briefly, the infected cells (16 h p.i.) were treated with 15 μg of ActD/ml for 1 h and then transfected with BrUTP (B 7166; Sigma) at a final concentration of 10 mM by using Lipofectamine 2000 (Invitrogen) and maintained in the presence of ActD for 20 min.

Techniques: Synthesized, Infection, Transfection, Marker, Incubation, Produced, Laser-Scanning Microscopy, Recombinant, Expressing, Labeling

Journal: Molecular Biology of the Cell

Article Title: Initiation of Nucleolar Assembly Is Independent of RNA Polymerase I Transcription

doi:

Figure Lengend Snippet: ActD at a concentration of 0.04 μg/ml selectively inhibits Pol I transcription without significantly affecting other polymerases and general nuclear organization. A and A′ show the in situ incorporation of Br-UTP, and B and B′ show the same cells corresponding to those in A and A′ immunolabeled with anti-fibrillarin antibody. Daughter cells grown in the presence of ActD form mininucleoli that are immunolabeled with anti-fibrillarin antibody (B, arrows). They do not incorporate Br-UTP (A, arrows), whereas untreated nucleoli (B′, arrows) actively incorporate Br-UTP after pulse labeling (A′). Although Br-UTP incorporation in nucleoli declines in treated cells, the nucleoplasmic Br-UTP incorporation from Pol II and Pol III transcription is not altered (A). The speckled distribution of SC35 (C) and the predominantly nuclear localization of hnRNP A1 (D) are also not changed significantly in treated cells. Bar, 10 μm.

Article Snippet: Antibodies used in this study included anti-UBF–specific human serum at a dilution of 1:300 ( ; ), anti-nucleolin mAb at a dilution of 1:3000 , anti-fibrillarin human serum at a dilution of 1:100 , anti-SC35 at a dilution of 1:1000 , anti-hnRNP A1 at a dilution of 1:300 , and anti-Br-UTP mAb at a dilution of 1:500 (Sigma Chemical, St. Louis, MO) for 1 h at room temperature.

Techniques: Concentration Assay, In Situ, Immunolabeling, Labeling

Journal: Advanced Science

Article Title: GLTSCR1 Negatively Regulates BRD4‐Dependent Transcription Elongation and Inhibits CRC Metastasis, GLTSCR1 Negatively Regulates BRD4‐Dependent Transcription Elongation and Inhibits CRC Metastasis

doi: 10.1002/advs.201901114

Figure Lengend Snippet: GLTSCR1 regulates transcriptional elongation and enhances CRC cell sensitivity to BET inhibitors A) Luciferase reporter assay to detect the transcriptional activity of SLC2A1 and SLC2A3 in GLTSCR1‐KO HCT116 cells. B) ChIP‐PCR to detect the DNA binding capacity of Flag‐GLTSCR1 to SLC2A1 and SLC2A3 gene in scramble + empty vector, scramble + Flag‐GLTSCR1, and shBRD4 + Flag‐GLTSCR1 cells through pull‐down by anti‐Flag. C) ChIP‐PCR to detect the DNA binding capacity of BRD4 to SLC2A1 and SLC2A3 gene in mock and GLTSCR1‐KO cells through pull‐down by anti‐BRD4. D) Detection for nascent RNA of SLC2A1 and SLC2A3 in mock and GLTSCR1‐KO cells through RNA pull‐down by anti‐BrUTP. E) Transcription efficiency of SLC2A1 and SLC2A3 in GLTSCR1‐KO HCT116 cells, as determined by RT‐qPCR. F) ChIP‐PCR to detect the RNA Pol II chromatin occupancy in multiple sites of SLC2A1 gene in mock and GLTSCR1‐KO cells through pull‐down by anti‐RNA Pol II (left) and to analyze the accumulation of RNA Pol II in gene body of SLC2A1 after releasing f rom DRB‐inhibition at 0, 20, and 40 min in mock and GLTSCR1‐KO HCT116 cells (right). The upper schematic diagram represents the primers sites of SLC2A1. G) ChIP‐PCR to detect the RNA Pol II chromatin occupancy in multiply sites of SLC2A3 gene in mock and GLTSCR1‐KO cells through pull‐down by anti‐RNA Pol II (left) and to analyze the accumulation of RNA Pol II in gene body of SLC2A3 after releasing from DRB‐inhibition at 0, 20, and 40 min in mock and GLTSCR1‐KO HCT116 cells (right). The upper schematic diagram represents the primers sites of SLC2A3. H) Relative mRNA expression (up) and Immunoblotting analysis (down) of SLC2A1 and SLC2A3 in HCT116 cells transfected with siSLC2A1 and siSLC2A3. I) Transwell assay to investigate the migratory and invasive properties of HCT116 cells transfected with siSLC2A1 and siSLC2A3. The histogram on the right shows the quantification analysis results. J) JQ1 and I‐BET inhibition efficiency in control (mock), GLTSCR1‐KO and GLTSCR1‐C7/C9 heterozygous mutated HCT116 cells. Data are presented as the mean ± SD; statistical significance was assessed by an unpaired t ‐test. * P

Article Snippet: 50 µL Magnetic beads (Dynabeads Protein G, 10004D, Invitrogen) and 10 µg (20 µL) of anti‐BrUTP monoclonal antibodies (BD Biosciences, 555627) and aliquot RNA were used to pull down the BrUTP‐labeled nascent RNA, which was ready for further RT‐qPCR.

Techniques: Luciferase, Reporter Assay, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Binding Assay, Plasmid Preparation, Quantitative RT-PCR, Inhibition, Expressing, Transfection, Transwell Assay

Journal: Molecular and Cellular Biology

Article Title: Nonphosphorylated Human La Antigen Interacts with Nucleolin at Nucleolar Sites Involved in rRNA Biogenesis

doi: 10.1128/MCB.24.24.10894-10904.2004

Figure Lengend Snippet: npLa accumulates in nucleoli and colocalizes with fibrillarin and newly synthesized RNA polymerase I transcripts. Normal human fibroblasts (HSC172) were incubated with AbNP and with either antifibrillarin (A to C) or anti-B23 (D to F). AbNP is represented in red (A and D), and antifibrillarin and anti-B23 (fluorescein isothiocyanate) are represented in green (B and E). Although these colocalizations were done in parallel, the images in panels A to C are intentionally shown at higher magnification than those in panels D to F. Panels A and B were overlaid to produce panel C, and panels D and E were overlaid to produce panel F. (G to I) Anti-BrUTP and npLa are visualized in the red and green channels, respectively, by confocal microscopy, and the red and green images were overlaid to visualize colocalization. BrUTP incorporation occurred in the presence of 0 (G), 2 (H), and 100 (I) μg of α-amanitin/ml.

Article Snippet: Cells were washed, permeabilized, and incubated for 10 min at 37°C in transcription buffer containing 0.5 mM 5-bromouridine 5′-triphosphate (BrUTP; Sigma) containing the appropriate concentration of α-amanitin and processed as described previously with primary monoclonal Ab against BrUTP (1:200; Caltag) and AbNP (1:50) in phosphate-buffered saline for 1 h, washed with phosphate-buffered saline, and incubated with secondary Ab conjugated with fluorescein or Texas red (Vector Laboratories) ( , ).

Techniques: Synthesized, Incubation, Confocal Microscopy