in each group. For the identification of Glut 2 expressing ductal cells, we performed double immunofluorescent staining for Glut 2 and insulin on pancreatic sections of 4-day-old rats. Sections adjacent to these double-stained sections were stained for CK20. Cell culture Human duct cell line Panc-1 used in this study was purchased from ATCC (LCG Promochem Molsheim, France). Panc-1 cells were grown in DMEM supplemented with 10% FBS and penicillin–streptomycin (1%) in a humidified atmosphere of 95% O2, 5% CO2 at 37uC. The cells were regularly seeded into T75 flask (VWR International, Fontenay-sous-Bois, France). Media were changed twice weekly. Inhibition of MAP kinases ERK1/2 pathway ERK1/2 pathway was inactivated by the inhibition of its specific upstream activator MEK1. MEK1 inhibition was performed both by using pharmacological inhibitor or by the inhibition of its translation by the means of specific antisense oligonucleotides. Cells were seeded in twelve-well plates and after 6 h culture with DMEM supplemented with 10% FBS, cells were changed to serum-free medium for 3 days, followed by appropriate treatments described below. Pharmacological inhibition of MEK1 was achieved by the use of specific inhibitor PD98059. Different doses (1, 10 and 20 mM) of PD98059 were tested. For pharmacological inhibition of MEK1, cells were pre-treated with the inhibitor 1 h before KGF treatment. Twenty four hours after addition of KGF, cells were processed for western blot analysis or for proliferation assay. To inhibit MEK1 translation, MEK1 antisense morpholino- oligonucleotides (Gene Tools LLC) (59-TGGGCGTCG- GCTTCTTCTTGGGCAT-39) were designed based upon the published rat and human common regions of MEK1 cDNA sequence [GenBankTM accession number NM 002755 (human) and NM 001008375 (rat)]. Antisense oligonucleotides were delivered using the DMSO endoporter delivery system as recommended by the manufacturer (Gene Tools LLC). Standard non-specific morpholinos (59-CCTCTTACCTCAGTTA- CAATTTATA-39) were used as control. Cells were incubated with MEK1 antisense morpholino oligonucleotides or with standard oligonucleotides (Std) for 24 h and then supplemented with KGF (30 ng/ml) for another 24 h. One hour before the end of the experiments, BrdU (10 mM) was added to the culture media. Incubations were terminated by the addition of lysis buffer for western blot analysis or by fixation in 4% formaldehyde for immunocytochemical analysis. Western Blotting Western blot analysis was performed to study phospho- and total- ERK1/2, MEK1, cyclin D1 and cyclin D2 protein expression. Briefly, 10 mg of protein were subjected to SDS-PAGE (10% acrylamide gel) and then transferred to a PVDF membrane for 2 h (120 V) using a Bio-Rad Mini Trans Blot electrophoretic transfer unit (Bio-Rad, Marnes-la-Coquette, France). The membranes were blocked for nonspecific binding with 5% nonfat dry milk in Tris- buffered saline (TBS, 20 mM Tris-HCl, 150 mM NaCl, pH 7.4) supplemented with 0.05% Tween 20 (TTBS) and then probed with the specific primary antibodies. After 3 washes with TTBS, membranes were incubated with appropriate horseradish peroxi- dase-conjugated secondary antibodies. Separated proteins were visualized by an ECL kit (GE Healthcare) and light emission was captured on X-ray film (GE Healthcare). Intensities of the respective KGF and Pancreatic Duct bands were examined by densitometric analysis (Scion Image Analyst program). Proliferation assay Proliferation assay was performed with BrdU incorporation method on Panc-1 cells using a Cell Proliferation Kit (GE Healthcare). BrdU was added to the culture media 1 h before the end of the experiments. Cells were fixed in 4% formaldehyde for 20 min then washed with PBS. Panc-1 cells were then stained for BrdU according to manufacturer’s instructions. Cells were then counterstained with hematoxylin. Statistical analysis Data are expressed as the mean values 6SEM. Comparisons between the groups were performed by ANOVA and post-hoc (Fisher) or Mann-Withney tests. p,0.05 was considered as significant. Figure 1. KGF stimulates cell proliferation and activates ERK1/2 pathway. (A) Panc-1 cells were cultured in serum-free medium with or without KGF (30 ng/ml) or with 10% FBS for 24 h. BrdU was added to the culture media 1 h before the end of the experiments. Results are expressed as mean values 6SEM of three independent experiments. ** p,0.01. (B) Phosphorylation of ERK1/2 was evaluated by western blot analysis in cell extracts from Panc-1 cells stimulated for 5, 20 and 60 minutes with KGF, or with 10% FBS for 20 minutes. Western blot analysis of total-ERK1/2 protein was used as control. doi:10.1371/journal.pone.0004734.g001 PLoS ONE | www.plosone.org 3 March 2009 | Volume 4 | Issue 3 | e4734