Fig. 5. S1P stimulates proliferation of dorsal pancreatic mesenchymal cells in a pertussis toxin-sensitive manner. (A-C) Primary cultures of 10.5 dpc wild-type dorsal pancreatic mesenchymal cells were established and incubated with or without 0.5 µM S1P or 0.2 µg/ml PTX (including a 2 hour pre-incubation)+0.5 µM S1P for 24 hours. Quantification of the total number of cells and the number of proliferating cells were estimated by counting DAPI- and BrdU-positive cells, respectively. Data are presented as fold increase over control. Asterisks indicate that S1P significantly increases cell proliferation and that PTX inhibits this effect, as determined by two-tailed t-test (P<0.05). Error bars represent s.e.m. To detect proliferating mesenchymal and endothelial cells, double immunofluorescence was performed with Isl1 (mesenchymal marker) and Ki67 (B), and CD31 (endothelial marker) and Ki67 (C). The ratio of mesenchymal and endothelial cells was estimated to be 9:1. Although both mesenchymal and endothelial cells underwent mitosis, the majority of the proliferating cells were of mesenchymal cell origin. (D,E) To detect changes in the migratory behaviour of the mesenchymal cells, F-actin was visualised by Phalloidin staining after a 24-hour incubation with (E) or without (D) 0.5 µM S1P. No change in F-actin rearrangement could be distinguished. Scale bars: in B, 20 µm for B,C; in D, 20 µm for D,E.