Nestin and Steroid Receptor Figure 11. Suppression of nestin by RNA interference induces a G1/S cell cycle arrest. (A, A1, A2) Human A172 cells transduced with nestin specific shRNA (Nes-shRNA) show loss of nestin expression (A) and increased nuclear GR staining (A1); (B, B1; B2) localization of Nes (B) and GR (B1) in A172 cells transduced with scrambled shRNA (scr-shRNA). (C) Cells expressing Nes-shRNA are arrested in G1/S transition, indicated by the altered cell cycle profile, the decrease in cyclin A expression, and maintenance of cyclin E expression (C, lane 3). (D) Suppression of GR in advance impairs activation of a G1/S arrest by Nes-shRNA. Cyclin A is detectable, cyclin E does not accumulate to high levels (D, lane 3). (E) Proportions of cell cycle phases in A172 cultures expressing scrambled, Nes specific, or in a sequential manner GR and Nes specific shRNA. Mean values and SDs are of three independent experiments, counting 10,000 cells in each experiment; bar in A and B = 10 mm. doi:10.1371/journal.pone.0006084.g011 with 5% fetal calf serum (FCS). Primary mouse cells prepared from Balb/C mouse embryos (E14.5) were kindly provided by Christina Heinlein, Heinrich-Pette-Institut. Hippocampal explants of rat embryos (E14.5) were kindly provided by M. Schweitzer, Center for Molecular Neurobiology, Hamburg, Germany; the explants were prepared as described by Neidhardt et al. [77]. Human peripheral blood mononuclear cells (PBMC) and human cord blood cells were isolated by Ficoll-Hypaque density centrifugation. Isolated cells were washed twice in RPMI 1640 and resuspended to give 16106 cells/ml in RPMI 1640 containing 10% FCS. PBMCs were incubated overnight at 37uC to separate adherent cells. Non adherent cells were collected and washed once with RPMI 1640 supplemented with 10% FCS. Antibodies, expression plasmids and transfections The following antibodies were used: mouse monoclonal anti- vimentin V9 and Vim3B4 (Boehringer Mannheim, Germany); goat anti-vimentin and rabbit anti-lamin B (P. Traub, Ladenburg, Germany); anti-glucocorticoid receptor M20 (sc-1004), E20 (sc- 1003) and H300 (sc-8992) (Santa Cruz Biotechnology, Santa Cruz, U.S.A.); anti-nestin R-20 (sc-21249) and C-20 (sc-21247) (Santa Cruz Biotechnology); mouse anti-tubulin (Oncogene Research, San Diego, U.S.A.), mouse anti-CD34 (BD Biosciences, Belgium), (mouse anti-CD133 (Mitenyi Biotec, Germany). Normal goat serum and normal donkey serum were purchased from DAKO (Glostrup, Danmark); donkey anti-mouse IgG, donkey anti-rabbit IgG, and donkey anti-goat antibodies conjugated with Alexa 488, 568, 555, or 633 (Molecular Probes, Leiden, Netherlands) were used as secondary antibodies. DRAQ5TM (Biostatus Limited, Leicestershire, England) was used to stain DNA. The expression vector for tailless vimentin was kindly provided by Peter Traub, Ladenburg, Germany. The nestin expression plasmid was kindly provided by J. Yang [78]. Immunocytochemistry Cells grown on glass coverslips were extracted in situ with pre- warmed 80 mM Pipes buffer pH 6.8 [1 mM MgCl2, 4 mM EGTA, 4% polyethylene glycol 6000 (PEG 6000), 1% Triton X- PLoS ONE | www.plosone.org 13 June 2009 | Volume 4 | Issue 6 | e6084