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