PKCdelta in Insulin Secreting Cells Figure 2. Nuclear-cytosolic distribution of the cell cycle inhibitor p21Cip1/WAF1 depends on PKCd in INS-1E cells. Representative Western blots of (A) p21Cip1/WAF1 and (B) p27kip1 expression in control, PKCdWT and PKCdKN INS-1E cells and means + SEM of n = 3 independent experiments of the amounts of p21Cip1/WAF1 and p27kip1 relative to GAPDH as determined by densitometry. * (p,0.05) represents significance to control INS-1E cells. (C) Subcellular distribution of p21Cip1/WAF1 protein as analyzed by confocal laser scanning microscopy. Nuclei are stained in red, p21Cip1/WAF1 in green. The percentage of p21Cip1/WAF1-positive nuclei is expressed as means + SEM of n = 3–4 independent experiments. ** (p,0.01) represents significance against control INS-1E cells. (D) Representative Western blots and relative quantities expressed as means + SEM of n = 3 independent experiments of p21Cip1/WAF1 in cytosolic and nuclear fractions of control and PKCdWT INS-1E cells. * (p,0.05) indicates significance to p21Cip1/WAF1 of control INS-1E cells set to 100%. doi:10.1371/journal.pone.0028828.g002 the inhibitor was apparent (Fig. 2C). The reduced nuclear localization of p21Cip1/WAF1 in PKCdWT cells was confirmed by Western blotting of cytosolic and nuclear fractions (Fig. 2D). In the cytosolic fractions the relative amount of p21Cip1/WAF1 was similar in control and PKCdWT cells. Furthermore, the cell cycle inhibitor remained cytosolic in PKCdWT cells also after synchronization of cells by serum removal (Fig. S2). Although an increased number of nuclei of PKCdKN INS-1E cells stained positive for p21Cip1/WAF1, the amount of p21Cip1/WAF1 protein detected on Western blots was not increased relative to the amount of nuclear proteins (data not shown). These findings do also support the hypothesis that PKCdWT cells proliferate faster due to reduced p21Cip1/WAF1 activity. To transfer this finding to native b-cells, proliferation of islet cells was examined in pancreatic slices of WT and b-cell specific PKCdWT transgenic mice. Even after high fat feeding, Ki67 staining was not detectable neither in WT nor in PKCdWT b-cells, which suggests that proliferation remained low (data not shown). However, p21Cip1/WAF1 immunoreactivity was found in nuclei of PLoS ONE | www.plosone.org 3 December 2011 | Volume 6 | Issue 12 | e28828