K-rasG12D induces ADM in explant culture.
Pancreatic acinar cells were isolated from LSL-Kras mice, incubated with adeno-Cre-GFP virus, and embedded in collagen (without exogenous TGF-α). A) Representative bright field and fluorescent images were captured on days 1, 3 and 7. GFP fluorescence indicates infection by adeno-Cre-GFP virus. Scale bar, 200 µm. B) ADM was confirmed by co-immunofluorescence of amylase (red) and CK-19 (green) in K-rasG12D–induced ductal cells on day 7. C) mRNA was isolated from day 1 and 6 explant cultures of Ad-Cre virus-treated LSL-Kras acinar cells and analyzed by qPCR for TGF-α expression. Data is presented relative to 18S abundance (×105) and is representative of two independent experiments. D) Pancreatic acinar cells were isolated from LSL-Kras mice, incubated with Ad-Cre and embedded in collagen ± 1 µM or 10 µM Erlotinib, or 10 µM NSC23766 for 5 days. Quantitative analysis of metaplastic duct formation is plotted for each treatment. Bars = mean ± SD. *P<0.05 (Student T-test). Plots are representative of two independent experiments. E) PKCι (red) was undetectable in LSL-Kras explant culture on day 1, but was elevated in K-rasG12D–induced ductal cells on day 7. Scale bar, 25 µm.