Activation of trypsinogen in large endocytic vacuoles of pancreatic acinar cells

Sherwood et al. 10.1073/pnas.0700951104.

Supporting Information

Files in this Data Supplement:

SI Movie 1
SI Figure 7
SI Figure 8
SI Figure 9
SI Figure 10

SI Figure 7

Fig. 7. The distribution of precleaved BZiPAR in pancreatic acinar cells. The pancreatic cell doublet was incubated in presence of BZiPAR and trypsin, which results in cleavage of BZiPAR and preferential staining of the basal part of the cells. (A) The transmitted light image of doublet of pancreatic acinar cells. (Scale bar: 10 mm.) (B) Distribution of fluorescence following BZiPAR cleavage by trypsin added to extracellular solution. (C) Confocal reflectance mode preferentially showing the distribution of secretory granules. (D) Combined image. Note that most of the staining that develops following BZiPAR cleavage is basal with respect to the localization of secretory granules.

SI Figure 8

Fig. 8. Relative distribution of endocytosed dextran and antibody staining for EEA1 in pancreatic acinar cells after stimulation with 10 nM CCK. (A) Shows transmitted light image of fixed and permeabilized pancreatic acinar cells. (Scale bar: 10 mm.) (B) The distribution of endocytosed Alexa Fluor 488 dextran (anionic-fixable). (C) Distribution of fluorescence of antibodies against EEA1. (D) Combined image showing that EEA1 fluorescence can be found close to the vacuoles but is not colocalizing with endocytosed material.

SI Figure 9

Fig. 9. Relative distribution of endocytosed dextran and antibody staining for LAMP1 after stimulation with 10 nM CCK. (A) Transmitted light image of fixed and permeabilized pancreatic acinar cells. (Scale bar: 10 mm.) (B) The distribution of endocytosed Texas red dextran (anionic lysine-fixable). (C) Distribution of fluorescence of antibodies against LAMP1. (D) Combined image showing partial colocalization of LAMP1 fluorescence with endocytic vacuoles. Structures showing colocalization are indicated by arrows; structures with no overlap of staining are indicated by arrowheads.

SI Figure 10

Fig. 10. GPN-mediated specific rupturing of lysosomes had no effect on endocytic vacuoles. Clusters of isolated pancreatic acinar cells were stimulated for 1 h with CCK (10 nM) in the presence of rhodamine green dextran (RGD, green). (Scale bar: 10 mm.) Lysosomes were labeled with the lysosomal marker Lyso-Tracker red, (Lyso-T-Red, red). Upper and Lower, respectively, show cells before (marked 1 on the graph) and after (marked 2 on the graph) GPN application. Treatment with GPN dispersed Lyso-Tracker red fluorescence (see red line in the graph, compare red staining in Upper with red staining in Lower). GPN had no effect on rhodamine green dextran labeling of large endocytic vacuoles (see green line in the graph, compare green staining in Upper with green staining Lower).

SI Movie 1

Movie 1. Formation and dynamics of large endocytic vacuoles. The movie illustrates the same experiment as shown in Fig. 1. A cluster of pancreatic acinar cells was incubated in the presence of Texas Red dextran. Addition of CCK (10 nM, at the time point shown by *) resulted in the formation of large endocytic vacuoles. The movie shows »80 min of recording.