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K-rasG12D induces ADM in explant culture.

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posted on 2012-02-16, 00:17 authored by Michele L. Scotti, Kristin E. Smith, Amanda M. Butler, Shelly R. Calcagno, Howard C. Crawford, Michael Leitges, Alan P. Fields, Nicole R. Murray

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.

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