cultured mouse and human islet cells (Fig. 3A and 3B). Similarly, mice islet cells with targeted expression of PKCdWT in b-cells showed reduced nuclear accumulation of p21Cip1/WAF1, whereas a prominent nuclear staining was evident in islet cells from control mice and PKCdKN transgenic mice (Fig. 3A). These observations suggest that PKCd-dependent regulation of p21Cip1/WAF1 might also influence proliferation of native b-cells. PKCd-dependent phosphorylation of p21Cip1/WAF1 at Ser146 regulates its subcellular distribution and function To investigate the molecular mechanism of PKCd-dependent subcellular distribution of p21Cip1/WAF1, the phosphorylation of p21Cip1/WAF1 at two regulatory sites was examined next. In comparison to control INS-1E cells phoshorylation of p21Cip1/WAF1 at Ser146 was significantly increased in PKCdWT cells, while it was reduced in PKCdKN cells (Fig. 4A). Phosphorylation of Thr145 was not detectable (Fig. 4B). To substantiate the effect of PKCd on p21Cip1/WAF1, its phosphorylation at Ser146 was examined in cells stimulated with the phorbol myristate acetate (PMA). When cells Figure 3. Expression of p21Cip1/WAF1 in mouse and human islets and PKCd-dependent changes in cytosolic-nuclear distribution in mouse islet cells. (A) Shown are representative pictures of immunocytochemical staining for p21Cip1/WAF1 in islet cells of control, PKCdWT and PKCdKN transgenic mice. Nuclei are stained in red, p21Cip/WAF1 in green. Note the absence of nuclear staining of p21Cip/WAF1 in PKCdWT mouse islet cells. (B) Immunohistochemical detection of p21Cip/WAF1 by laser scanning microscopy in cultured human islet cells using two distinct antibodies from Cell Signalling (sc Ab) and BD Biosciences (BD Ab). doi:10.1371/journal.pone.0028828.g003 PKCdelta in Insulin Secreting Cells were starved overnight, phosphorylation at Ser146 declined (Fig. 4C, first and second line). PMA stimulated p21Cip1/WAF1 phosphorylation in starved cells, an effect that was completely abolished by the PKCd inhibitor rottlerin. Inhibition of phosphorylation was accompanied by an increase in p21Cip1/WAF1 protein (Fig. 4C). Moreover, the effects were specific for PKCd, as inhibition of protein kinase B or ERK1/2 neither inhibited phosphorylation at Ser146 nor promoted nuclear accumulation of p21Cip1/WAF1 (Fig. S3). As a matter of fact nuclear staining of p21Cip1/WAF1 was reduced after stimulation of PKCs with PMA and was increased after treatment of INS-1E cells with rottlerin (Fig. 5). Similar but less pronounced results were obtained with PKCdWT INS-1E cells (data not shown). Although rottlerin is a sensitive inhibitor of PKCd, it also affects other kinases such as CaM kinase III [35]. Therefore, we used the more specific siRNA- approach to reduce PKCd expression and activity. In cells transfected with siRNA against PKCd a significant reduction of PKCd expression was accompanied by a reduced phosphorylation at Ser146 and a concomitant increase in the protein amount of p21Cip1/WAF1 (Fig. 6A), an effect not found in cells treated with control siRNA. Consistent with the results obtained with PKCdKN cells, the knockdown of PKCd by siRNA resulted in an increased nuclear translocation of p21Cip1/WAF1 (Fig. 6B). These data strongly suggest that p21Cip1/WAF1 is a substrate of PKCd in insulin-secreting cells. Phosphorylation of p21Cip1/WAF1 by PKCd results in its nuclear extrusion and thereby may support proliferation. Functional consequences of reduced PKCd expression in insulin-secreting cells under stress-free conditions As PKCd supports fatty acid induced apoptosis, the effect of changes in PKCd expression on cell death was examined in more detail. Surprisingly, inhibition of endogenous PKCd with siRNA or with PKCdKN mutant almost doubled the incidence of apoptotic cell death under non-stress conditions, as revealed by TUNEL staining (Fig. 6C and Fig. 6D). In accordance, isolated islet cells from transgenic mice expressing PKCdKN in b-cells displayed increased TUNEL staining when compared to control mice (Fig. 6E). In contrast, overexpression of PKCdWT in INS-1E cells and mice b-cells did not stimulate apoptosis under control culture conditions (Fig. 6D and 6E). These observations suggest that PKCd per se is not pro-apoptotic but rather promotes proliferation. Finally, the impact of changes in PKCd expression on cell cycle was examined. When cells were stained for the G2/M marker phospho-Ser10 histone H3, the same amount of control and PKCdWT nuclei (7%), but significant more PKCdKN nuclei (17%) stained positive for phospho-Ser10 histone H3 (Fig. S4). Cell cycle analysis by flow cytometry of propidium iodide-stained nuclei revealed two distinct DNA peaks (Fig. S5A). While the major peak of control cells (58%) and significant more PKCdWT INS-1E cells (70%) showed similar DNA staining which represents G1 (2n chromosomes), a minor part of the cells resided in G2 (4n). The first DNA peak of PKCdKN cells was visible at 4n (80%) and the second peak at 8n (20%), which probably represent cells with increased DNA content at G1 and G2, respectively. That the DNA peaks correlate to 2n, 4n and 8n was confirmed by treatment of the cells with colchicine (0.5 mM for 2 d) which arrests cell cycle at G2/M transition (Fig. S5B). When DNA from freshly isolated mouse islet cells was examined, more than 95% of WT and PKCdKN cells stained for 2n and less than 2% of the cells for G2 (Fig. S6). The prominent peak (2n) of WT and PKCdKN islet cells suggests that mouse islet cells are arrested in G0/G1 and that PKCdKN expression did not affect the arrest. PLoS ONE | www.plosone.org 4 December 2011 | Volume 6 | Issue 12 | e28828