Fig 2: Cftr is functionally detected in rat ß-cells.A. Representative Cl-currents recorded using rat primary pancreatic ß-cells pre-incubated for 1h in 5.5 or 12.5mM glucose before (control, black trace), after forskolin addition (10µM, blue trace) alone or 5 min after adding CFTRinh-172 (5µM, red trace). B, C. Current density-voltage relationships of ß-cell Cl-currents recorded in 5.5mM (n = 10) glucose (B) or 12.5mM (n = 8) glucose (C) in the presence of forskolin (10µM, blue squares) alone or plus CFTR-inh-172 (5µM, red squares). Insets in B and C denote peak currents at +100mV (n = 8, for all conditions, *p<0.05). D. Current density-voltage relationship of ß-cell Cl-currents in 5.5mM or 12.5 mM glucose after addition of forskolin (10µM). Traces show ß-cell responders (blue circles/squares, n = 10 and n = 8 for 5.5mM and 12.5 mM respectively) and non-responders to forskolin in 5.5mM or 12.5mM glucose (open triangles, n = 19 and n = 41, respectively). E. Numerical proportion of the latter observation.

Fig 3: Cftr is expressed in endocrine cells of the mouse islet.A-J. Immunofluorescence images of WT mouse (CftrWT) pancreas immunostained with the Cftr antibody acl-006 [(Table 1), A and F] in combination with antibodies against insulin (Ins, B), glucagon (Glc, C), somatostatin (Stt, H) and pancreatic polypeptide (PP, G). Magnified overlay images of D and I are shown in E and J, respectively, wherein Cftr immunoreactivity in a-, ß-, d- and PP-cells of the mouse islet is indicated by arrows. K-L. Immunohistochemistry images of normal human pancreas immunostained with acl-006. The islet encircled by a dashed red trace in K is magnified in L to show CFTR immunoreactivity. Bars in A-G represent 25µm.

Fig 4: CFTR antibody validation.A-D. Shown are microscopy images of human pancreas from a normal donor (A, B, 11-month-old control) or homozygous for the CFTR mutation F508del (C, D, 6-month-old CF patient) immunostained using CFF-217 (A, C) or CFF 412 (B, D). Note the expected apical and intracellular immunolabeling of CFTR in normal and mutant pancreas, respectively. The insets indicate higher magnification. E, F. Microscopy images of human pancreas from a 1 yr. old CF patient homozygous for CFTRG542X using the antibodies CFF-217 (E) and CFF-412 (F). Shown is absence of immunostaining with both antibodies. G-I. Microscopy images of pancreas (G) and intestine (H) of CftrKO mice immunolabeled using CFF-217. Shown are the expected patterns of immunostaining for specific antibodies. For comparison, shown are magnified images of CftrWT pancreas islets using CFTR and insulin antibodies (top and bottom right corners in G, respectively). I. Omission of the CFTR antibody did not generate immunostaining in the intestine. Bars in A-I represents 50µm. J-L. Pancreatic tissue from CftrG542X mice co-immunolabeled by using the indicated mouse CFTR-specific antibody acl-006 (J), insulin (K, Ins) and glucagon (L, Glc). M-N. Overlay image of J-L and magnified squared area shown in N. O-P. Control images obtained from CftrG542X mice in the absence of primary (O) or secondary antibodies (P). DAPI was used to counterstain nuclei in P.

Fig 5: Cftr contributes to the insulin secretory response in mouse, rat and human islets and the MIN6 ß-cell line.A. Proof of specificity for CFTRinh-172 (0–10µM) on the secretory response using islets obtained from transgenic mice lacking Cftr (CftrKO) and CftrWT mice, in response to 5.5mM and 12.5mM glucose (n = 8, *p<0.05). B, C. Effect of 5µM CFTRinh-172 on insulin secretion of mouse CftrKO and CftrWT islets (B, n = 5, *p<0.05) and rat islets (C, n = 3, *p<0.05) in response to 5.5mM and 12.5mM glucose. D. Basal (5.5mM glucose) and stimulated (12.5mM glucose) insulin secretory response of freshly isolated primary human islets in the presence of vehicle (DMSO) or 5µM CFTRinh-172 (n = 5 donors, *p<0.05). E. Dose-response curve of basal (5.5mM glucose) and stimulated (12.5mM glucose) insulin secretion from human islets (n = 4 donors, *p<0.05) treated with the indicated concentrations of CFTRinh-172. F. Basal (5.5mM glucose) and stimulated (12.5mM glucose) insulin secretory response of MIN6 ß-cells incubated with vehicle (DMSO) or 5µM Inh172 (n = 3, *p<0.05).