Figure 3: PKD1 is necessary for Kras-induced metaplasia to ductal structures.
(a) Pancreatic tissue (normal acinar area and regions with ADM) from bi-transgenic p48cre;KrasG12D mice was stained with haematoxylin and eosin (H&E), Alcian Blue, or analysed by IHC for PKD1 expression (anti-PKD1). The asterisk shows a typical region of newly formed ductal structures. The arrow shows immune cells staining positive for PKD1. Scale bar, 50 μm. (b,c) Primary acinar cells were isolated from LSL-KrasG12D mice and infected with Adeno-Cre (or control virus) to induce expression of KrasG12D. Formation of ductal structures was quantified and ducts formed were photographed (b), or ducts formed were isolated and analysed by western blotting for PKD1 expression (anti-PKD1) or activity (anti-pS744/748) and anti-amylase (c). In b, scale bar, 200 μm; in c, silver staining served as loading control. (d) Primary acinar cells from LSL-KrasG12D mice were isolated from mouse pancreas and infected with lentivirus harbouring (scrambled) control shRNA (Ctrl-shRNA) or two different sequences of PKD1-shRNA (#1 and #2) specifically targeting mouse PKD1. Expression of mutant Kras was induced by adenoviral infection of cre-recombinase as indicated (Adeno-Cre), or Adeno-null as control. In addition, cells were lentivirally infected with human PKD1 or control virus as indicated. Cells were then seeded in 3D culture in collagen and formation of ductal structures was quantified. (e) Primary acinar cells were isolated from mouse pancreas, lentivirally infected with KrasG12V and then seeded in 3D culture in collagen in the absence or the presence of the PKD inhibitor kb-NB-142-70 at indicated doses. Formation of ductal structures was quantified. * Statistical significance (P<0.05; Student’s t-test) as compared with control. ** Statistical significance (P<0.05; Student’s t-test) as compared with stimulus. *** Statistical significance (P<0.05; Student’s t-test) as compared with respective shRNA sample. Error bars (s.d.) were obtained from three experimental replicates. (f) Analysis of control, p48cre;KrasG12D and p48cre;KrasG12D;PKD1−/− mice for regions of ADM/PanIN using H&E staining and IHC for claudin-18. Additional controls are depicted in Supplementary Fig. 3. Scale bar, 50 μm. (g) Quantitation of ADM and PanIN lesions in p48cre;KrasG12D and p48cre;KrasG12D;PKD1−/− mice.
