Figure 2: PKD is necessary for TGFα-mediated metaplasia to ductal structures.

(a,b) Primary acinar cells 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. In addition, cells were lentivirally infected with human PKD1 or control virus, as indicated. Cells were then seeded in 3D culture in collagen and transdifferentiation was induced with TGFα (50 ng ml⁻¹). Formation of ductal structures was quantified (a), or ducts formed were photographed and the ductal area of 100 ducts was determined (b). In b, scale bar, 200 μm. (c) Primary acinar cells were isolated from mouse pancreas, seeded in 3D culture in collagen and transdifferentiation was induced with TGFα (50 ng ml⁻¹) in the absence or the presence of the PKD inhibitor kb-NB-142-70 at indicated doses. ADM events were quantified. (d) Primary acinar cells were isolated from mouse pancreas and stimulated with TGFα (50 ng ml⁻¹, 48 h). Active Ras was pulled down using GST-Raf-RBD. Samples were analysed by SDS–PAGE and immunoblotting for pulled-down active Ras (anti-Ras), GST-Raf-RBD input (anti-GST), as well as other input controls (anti-Ras and anti-β-actin). (e) Primary acinar cells were isolated from mouse pancreas, infected with lentivirus harbouring (scrambled) control shRNA (Ctrl-shRNA) or Kras-shRNA (two different specific sequences, #1 and #2), seeded in 3D culture in collagen and transdifferentiation was induced with TGFα (50 ng ml⁻¹). ADM events were 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 TGFα treatment. *** 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. All experiments shown were performed at least three times with similar results.