KGF and Pancreatic Duct Figure 3. Inactivation of ERK1/2 pathway by antisense morpholino-oligonucleotides abolishes KGF-induced proliferation of Panc-1 cells. (A) Panc-1 cells were cultured in serum-free medium and treated for 24 h with MEK1 antisense morpholino-oligonucleotides (MEK1-AS, 10 nmol/ml) or standard morpholino-oligonucleotides (Std, 10 nmol/ml). Cell lysates were analyzed by western blot using specific antibody against MEK1. Representative blots and MEK1 protein quantification are shown in Figure 3A. Results are expressed as mean values 6SEM of 4 independent experiments. * p,0.05. (B) Panc-1 cells were treated with MEK1-AS or Std during 24 h, then stimulated with KGF for 5 minutes before the end of the experiments. Cell lysates were analyzed by western blot for P-ERK1/2 and total-ERK1/2 expression. (C) Panc-1 cells were cultured in serum-free medium and pre-treated with MEK1-AS during 24 h prior the addition of KGF or 10% FBS. BrdU labelling index of Panc-1 cells was evaluated and expressed as the percentage of BrdU-positive Panc-1 cells. Results are presented as mean values 6SEM of three independent experiments. * p,0.05. doi:10.1371/journal.pone.0004734.g003 cell proliferation. However, because of the fundamental intercon- nectedness of signaling networks, it cannot be ruled-out that interactions with proteins beyond the one intended could be caused by small kinase inhibitors [12,13]. To definitely exclude the possibility of non-specific effects of PD98059, we used a specific approach to knock down MEK1 protein by antisense morpholino-oligonucleo- tides. Morpholino-oligonucleotides are useful tools to silence gene expression post-transcriptionally [14,15]. We first verified that morpholino-oligonucleotides successfully penetrated into the cells by the use of FITC-conjugated MEK1-AS (not shown). Then we showed that the expression of MEK1 protein was decreased by about 40% in Panc-1 cells treated with MEK1-AS (Figure 3A). Downregulation of MEK1 by specific antisense oligonucleotides blunted ERK1/2 phosphorylation (Figures 3B). KGF-induced cell proliferation was completely abolished in Panc-1 cells treated with MEK1-AS (Figures 3C). Induction of cell cycle regulators by KGF. We evaluated the expression of D-type cyclins (D1 and D2) in Panc-1 cells by western blot analysis. Cyclin D2 was not expressed in Panc-1 cells (Figure 4A) whereas cyclin D1 was expressed at a low level in serum deprived Panc-1 cells. We analyzed the effect of KGF on the expression of cyclin D1, 8 h after incubation with the peptide. KGF significantly increased the levels of cyclin D1 protein in the cells (Figure 4B). Blockade of ERK1/2 pathway by PD98059 silenced the KGF-induced expression of cyclin D1 indicating that induction of this protein by KGF is ERK1/2 dependent (Figure 4B). II- In vivo studies Activation of ERK1/2 pathway by KGF. We first investigated whether KGF induces ERK1/2 activation in the pancreas of neonatal diabetic rats. We found a significant increase of phosphorylated ERK1/2 in the whole pancreatic extracts of 2- PLoS ONE | www.plosone.org 5 March 2009 | Volume 4 | Issue 3 | e4734