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: 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

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