Human islet preparations were obtained from the ECIT (European Consortium of Islet Transplantation) Center in Geneva (Switzerland). Human islets, purified by hand picking, were digested to single cells with trypsin (40 units/ml trypsin-EDTA in PBS) for 4–6 min at 37uC. Isolated islet cells were then cultured in CMRL 1066 medium (GIBCO, Invitrogen GmbH, Karlsruhe, Germany) containing 5.5 mM glucose, 10% fetal calf serum (FCS, Biochrom, Berlin, Germany), 2 mM L-glutamine and 10 mM HEPES. After 2 d culture on collagen (2 mg/ml human collagen type 1) coated glass cover slips cells were used for immunocyto- chemical staining. Transgenic mice were generated and isolated islet cell culture was prepared as described previously [14,53]. Cell cycle analysis by Nicoletti After 2 d culture, cells were detached by trypsin and resuspended in Nicoletti buffer containing 0.1% sodium citrate, pH 7.4, 0.1% Triton X-100 and 50 mg/ml propidium iodide. DNA staining was analyzed by flow cytometry using the FL2-H channel. Immunocytochemistry and TUNEL staining Isolated mouse islet cells, INS-1E cells and MEFs were cultured for 2 d on L-poly-ornithine (0.001%) coated glass cover slips. Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), permeabilized with 0.2% Triton X-100 and preincubated in 10% FCS-PBS for 45 min. Primary antibodies against phospho-Ser10 histone H3 (1:150, from Millipore, Billerica, MA), p21Cip1/WAF1 (rabbit polyclonal antibody, 1:200, from Santa Cruz Biotechnology, Santa Cruz, CA or mouse monoclonal antibody, 1:150, from Becton Dickinson, Heidelberg, Germany), Ki67 (1:50, DakoCytomation, Hamburg, Germany) were applied overnight in 10% FCS-PBS. After 30 min washing with FCS-PBS the cells were incubated for 1 h with the secondary antibody in 10% FBS-PBS (Alexa-Fluor488 coupled anti-rabbit or anti-mouse IgG, 1:400, Invitrogen GmbH, Darmstadt, Germany). Thereafter, nuclei were stained with 1 mM TOPRO-3 (Invitrogen) in PBS for 1 h. The fluorescence was examined with a confocal laser scanning microscope (Leica, Wetzlar, Germany). For TUNEL staining cells were prepared according to the protocol provided by the commercial kit (Roche Diagnostics, Mannheim, Germany). Western blotting Islets and INS-1E cells were lysed in buffer containing 125 mM NaCl, 1% Triton X-100, 0.1% SDS, 10 mM EDTA, 25 mM HEPES pH 7.3, 10 mM NaPP, 10 mM NaF, 1 mM Na-vanadate, 10 mg/ml pepstatin A, 10 mg/ml aprotinin and 0.1 mM PMSF. Protein concentrations of cell lysates were determined using the Bradford dye-binding procedure from Biorad Laboratories (Munich, Germany). Cytosolic and nuclear fractions of INS-1E cells were prepared using a commercial kit (Pierce Biotechnology, Rockford IL). Cell homogenates or cytosolic and nuclear fractions were subjected to SDS-PAGE (8–12%) and blotted onto nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). Membranes were incubat- ed overnight with primary antibodies (diluted 1:1000 in TBS-Tween containing 5% BSA or 5% milk powder) followed by incubation with horseradish peroxidase-coupled anti-rabbit IgG (1:2000 in TBS- Tween, 5% milk powder). Antibodies against PKCd, phospho- Thr505 and phospho-Ser643 PKCd, p27 and tubulin were from Cell Signaling Technology (Danvers, MA), antibodies against GAPDH, histone H1, and polyclonal antibodies against p21Cip1/WAF1 and phospho-p21Cip1/WAF1 (phospho-Ser146, phospho-Thr145) were from Santa Cruz Biotechnology. PKCdelta in Insulin Secreting Cells Statistics Data are expressed as means 6 SEM, p,0.05 (unpaired Student’s t-test or 2-way ANOVA followed by Bonferroni post test where applicable) was considered significant. Supporting Information Figure S1 Specificity controls of the p21Cip1/WAF1 anti- bodies. (A) Shown is a Western blot of homogenates from p21Cip1/WAF1 proficient or deficient MEFs that were either left untreated or incubated with the DNA-damaging agent bleomycin (BLM, 10 mM for 20 h). On Western blots p21Cip1/WAF1 migrates at an apparent molecular weight of 17–18 kD. Tubulin was used as loading control. (B) Immunocytochemical staining of p21Cip1/WAF1 in p21Cip1/WAF1-proficient and deficient MEFs cultured under control conditions (con) or in the presence of bleomycin (BLM) using antibodies from Santa Cruz (left pictures) or Becton Dickinson (BD, right pictures). (TIF) Figure S2 Cell cycle dependent expression of p21Cip1/WAF1. Shown are representative pictures of immunocytochemical staining for p21Cip1/WAF1 (A) 16 h after serum deprivation and (B) 32 h after re-addition of 10% serum in control, PKCdWT and PKCdKN INS-1E cells. Nuclei are stained in red, p21Cip/WAF1 in green. Note the absence of nuclear staining of p21Cip/WAF1 in PKCdWT INS-1E cells 32 h after re-addition of 10% serum. (TIF) Figure S3 Phosphorylation and nuclear extrusion of p21Cip1/WAF1 is not mediated by PKB/Akt or ERK1/2. (A) Western blot analysis representative for 3 independent experi- ments with PKCdWT cell homogenates for the status of Ser146 p21Cip1/WAF1 phosphorylation. Cells were cultured for the indicated time in the presence of the protein kinase B inhibitor Akti-1/2 (Akti, 5 mM) or PD98059 (PD, 10 mM), a specific inhibitor of the ERK upstream MEK kinases. (B) Immunocyto- chemical staining for p21Cip1/WAF1 (green) in PKCdWT cells that were either left untreated or incubated for 24 h in the presence of Akti-1/2 (5 mM) or PD98059 (10 mM). Nuclei are stained in red. Both inhibitors (Akti and PD98059) were effective even after prolonged cell culture. Thus, IGF-1-induced PKB phosphoryla- tion was inhibited in the cells treated with Akti. Phorbol ester- induced phosphorylation of ERK and c-fos induction were inhibited in the cells treated with PD98059 (data not shown). (TIF) Figure S4 Changes in cell cycle progression of INS-1E cell expressing PKCdKN. Representative pictures of immuno- cytochemical staining for phospho-Ser10 histone H3. Nuclei are stained in red, phospho-Ser10 histone H3 in green. The percentage of positive cells is given as means 6 SEM from 3–4 independent experiments. * (p,0.05) represents significance to control INS-1E cells. (TIF) Figure S5 Cell cycle analysis of INS-1E cells. Represen- tative FACS measurements of propidium iodide-stained nuclear DNA from control INS-1E, PKCdWT and PKCdKN cells (A) after standard culture and (B) after treatment with colchicine (0.5 mM for 2 d) Results show means + SEM from n = 3–4 independent experiments. * (p,0.05) and ** (p,0.01) represent significance to the respective cell cycle phase of control INS-1E cells; ## (p,0.01) represents significance to the respective condition without colchicine treatment. (TIF) PLoS ONE | www.plosone.org 9 December 2011 | Volume 6 | Issue 12 | e28828