Nestin and Steroid Receptor Figure 2. Human PBMCs anchoring GR express nestin and the stem cell marker CD133. (A–D) Confocal images of PBMCs isolated from cord blood; (A1–A3) cells were attached to glass coverslips, fixed with acetone, and double labelled with antibodies to GR and Nes. The proportion of Nes positive cells is less than 1%. (B) Nes positive cells are negative for the hematopoietic lineage marker CD34.(C) Nes positive cells express the stem cell marker CD133 (merged image). (D) Merged image of cells extracted with a non-ionic detergent, fixed with paraformaldehyde, double labelled with antibodies to GR and Nes and incubated with the DNA stain DRAQ5 [79]. Cytoplasmic GR in Nes expressing cells resists extraction with a non- ionic detergent, whereas Nes negative cells retain GR only in the nucleus (arrow). Bars in B, C, and D = 5 mm. doi:10.1371/journal.pone.0006084.g002 C6D8 cells is insoluble, and the minor amounts which could be released with the mild extraction conditions were associated with GR. To substantiate the relationship between presence of nestin and vimentin, and retention of GR in the cytoplasm we depleted nestin from C6D8 cells by use of specific siRNA. As shown in Figure 6A, siRNA transfected cultures showed areas where cells were nestin negative (marked by a white line). GR became exclusively nuclear in these nestin depleted cells and the cells flattened and enlarged. No such changes were obtained by transfection with a scrambled siRNA (Figure 6B). In another approach we expressed an exogenous nestin protein in rat1 cells. Rat 1 cells contain a vimentin filament system but completely lack nestin and show GR exclusively in the nucleus (Figure 6C). When rat1 cells were transfected with a nestin expression vector, GR accumulated in the cytoplasm (Figure 6D). The data indicate that co-expression of nestin and vimentin is required to provide a structural basis for cytoplasmic anchoring of GR. Further to substantiate the association of GR with IFs we asked whether altering the structure of the vimentin filament network would induce a coincident rearrangement of GR. We transfected the vimentin deficient C6D10 cells with an expression vector coding for a C-terminal truncated vimentin protein (ct-vimentin). Although not essential for the polymerization itself the tails seem to stabilize the lateral interaction of vimentin filament subunits. In addition, they seem to mediate the structural interaction of vimentin with other major cytoskeleton filament systems [34]. As shown in Figure 7A–C tailless vimentin was still capable of forming a filament network, but this was composed of shortened filaments and less well organized. GR colocalized with these PLoS ONE | www.plosone.org 4 June 2009 | Volume 4 | Issue 6 | e6084