Nestin and Steroid Receptor Figure 6. Cytoplasmic localization of GR depends on the presence of nestin. (A) Suppression of Nes expression in C6D8 cells by transfection with nestin specific siRNA abolishes cytoplasmic GR staining; only nuclear GR staining is left. (C) GR is only nuclear in rat 1 cells which contain vimentin, but lack nestin. (D) Ectopic expression of nestin in rat1 cells induces cytoplasmic accumulation of GR. Bars in A, B, C, F, and G = 5 mm. doi:10.1371/journal.pone.0006084.g006 cells (Figure 10G). The data indicate that the non-activated form of GR identified by reaction with the phospho-Ser226 antibody is retained on the cytoskeleton. Suppression of nestin induces nuclear localization of GR and a sustained growth arrest In late passages of MEFs, when the cells had ceased proliferation and adopted a senescence like phenotype, nestin positive cells were hardly detectable. This let us ask whether loss of nestin could be causally related to activation of growth suppression. To evaluate this hypothesis we assayed the impact of nestin loss on cell growth in A172 cultures, a human cell line expressing nestin and vimentin and showing cytoplasmic anchoring of GR to this filament system. Nestin was suppressed by transduction of the cells with nestin specific shRNA (compare Figure 11, A and B). Suppression was obvious in Western-blot already 24 h post transduction (data not shown). Concomitantly, in immunofluorescence the nuclear GR signal increased relative to the cytoplasmic signal (compare Figure 11, A1 and B1). Flow cytometry analysis showed an increase in the proportion of G1 cells in cultures transduced with nestin shRNA, whereas the proportion of S-phase and G2 phase cells declined (Figure 11, C and E). The cyclin pattern revealed a decrease in the cyclin A level in nestin shRNA transduced cells, whereas the cyclin E level was maintained or even increased (Figure 11C, lane 3). Nestin shRNA transduced cells could not be subcultivated in contrast to cells transduced with a scrambled (scr) shRNA. It indicates that suppression of nestin arrested A172 cells irreversibly in G1/S. To determine if growth suppression induced by loss of nestin required GR, we depleted cells of GR by use of shRNA, and subsequently transduced these cells with nestin specific shRNA. Although GR and nestin were suppressed (Figure 11D, lane 3), cyclin A was still detectable, cyclin E stayed at a lower level than in cells transduced with nestin shRNA alone, and a significant proportion of cells were in S-phase (Figure 11E). This indicates that cells depleted of GR were less efficiently arrested in G1/S upon suppression of nestin, compared to PLoS ONE | www.plosone.org 8 June 2009 | Volume 4 | Issue 6 | e6084