Spatial Segregation of BMP/Smad Signaling Figure 2. Smad1/5/8 nuclear translocation is delayed and transcriptional activity is reduced by inhibition of dynamin-dependent endocytosis. (A) Serum-starved C2C12 cells were treated for 30 min with 40 mM dynasore or 0.05% DMSO prior to stimulation with 10 nM BMP-2 for 30 min in medium containing dynasore or DMSO. After fixation, endogenous Smad1 was stained using a specific antibody, nuclei were stained by Hoechst dye, and cells were analyzed by fluorescence microscopy. The panels shown are representative of two independent experiments. Bar, 10 mm. (B) Quantification of the experiment shown in (A). Relative fluorescence intensity of nuclear to cytoplasmic Smad1 staining is depicted in the histogram. The results are mean 6 s.d. of at least 100 cells. AU, arbitrary units. (C) Serum-starved C2C12 cells were treated for 2 h with 40 mM dynasore or 0.05% DSMO prior to stimulation with 10 nM BMP-2 for the indicated time periods in medium containing dynasore or DMSO. Samples were subjected to cytoplasmic-nuclear fractionation and processed for immunoblotting with anti-phospho-specific Smad1/5/8 antibody (anti- pSmad1/5/8). To control fractionation, samples were analyzed using anti-GAPDH or anti-Histon H3 antibodies. The Western Blot is representative of two independent experiments. c, cytosol; n, nucleus. (D,E) C2C12 cells were co-transfected with BRE-Luc and RL-TK. Cells were serum-starved and treated with 40 mM dynasore or 0.05% DMSO for 1 h prior to stimulation with 3 nM BMP-2 for 6 h (D) or 24 h (E) in medium containing dynasore or DMSO. Relative luciferase activity (RLA) of BRE-driven luciferase compared to constitutive expression of RL-TK is shown. Results are mean 6 s.d. of triplicate measurements, representative of three independent experiments. doi:10.1371/journal.pone.0025163.g002 target genes of BMP signaling [16]. Interestingly, only Id1 and Id3 were inhibited by dynasore (Figure 4A), whereas Id2 was not affected (Figure 4B). The difference between Id1 and Id2 gene expression in response to BMP-2 stimulation under endocytosis inhibition conditions was also confirmed in reportergene assays with reporter constructs containing the Id1 or Id2 promoter regions (Figure 4C) [17,18]. Furthermore, Dlx2 and Dlx3 belong to the family of Distal-less homeobox genes and are important players in organ development [19]. Both proteins are known early target genes of BMP signaling in C2C12 cells [20]. Similar to the Id-family of proteins, Dlx2 and Dlx3 are differently affected upon inhibition of endocytosis, although they belong to the same protein family. Thus, BMP-2-stimulated Dlx2 gene expression is inhibited by dynasore, whereas Dlx3 is unaffected (Figure 4A,B). Hey1 belongs to the hairy/Enhancer of split-related repressor protein basic helix-loop-helix family and is a direct BMP target gene [21]. Hey1 expression upon BMP-2 addition is also affected by inhibition of dynamin-dependent endocytosis, resembling Id1, Id3 and Dlx2 (Figure 4A). In contrast, Krt16 and Zbtb2 expression was not affected by dynasore treatment during BMP-2 stimula- tion. Both proteins have not been described yet to be target genes in BMP signaling. Krt16 belongs to the type I keratin family of proteins and was shown to be involved in keratinocyte migration [22]. The zinc finger and BTB domain-containing protein 2 (Zbtb2) is described as a transcription factor that represses p53 function [23]. In addition to the microarray data, these results further support the notion that two classes of BMP target genes exist, which are differentially affected by inhibition of dynamin- dependent endocytosis and thus by altered Smad1/5/8 phos- phorylation kinetics. PLoS ONE | www.plosone.org 4 October 2011 | Volume 6 | Issue 10 | e25163