Research Design and Methods

SUPPLEMENTAL DATA

RESEARCH DESIGN AND METHODS

siRNA and shRNA sequences- siRNA for mouse IGF-1R (sense, 5’-CGGAUUGAUCCUAAUGUAUtt-3’; antisense, 5’-AUACAUUAGGAUCAAUCCGtt-3’) (siRNA ID# 159116) and siRNA for mouse IR (sense, 5’-GGUGAGAAGACCAUUGAUUtt-3’; antisense, 5’-AAUCAAUGGUCUUCUCACCtt-3’) (siRNA ID# 67808) were purchased from Ambion (Cambridgeshire, UK). siRNA for Luciferase used as unrelated siRNA(sense, 5’-CGUACGCGGAAUACUUCGAtt-3’; antisense, 5’-UCGAAGUAUUCCGCGUACGtt-3’) (#1022070) was from Qiagen (Hombrechtikon, Switzerland). shRNA for mouse IGF-2 (sense, 5’-GCTTGTTGACACGCTTCAGTT-3’; antisense, 5’-AACTGAAGCGTGTCAACAAGC-3’) (clone ID# TRCN0000071148) and unrelated shRNA for mouse IGF-2 (sense, 5’-GCTTCTACTTCAGCAGGCCTT-3’; antisense, 5’-AAGGCCTGCTGAAGTAGAAGC-3’) (clone ID# TRCN0000071152) were in the pLKO.1 vector and were purchased from BioCat (Heidelberg, Germany).

For apoptosis assay, MIN6 cells were seeded at 200,000 cells in 12 well plates and were transfected one day after plating with 0.5 µg of a GFP-expression plasmid and 1.1 µg of IGF-2 shRNA or unrelated shRNA using lipofectamine 2000 (Invitrogen, Carlsbad, CA). After 6 hours at 37°C the growth medium was replaced. The next day, apoptosis was induced by exposing the MIN6 cells to cytokines at the indicated concentrations for 24 hours; GLP-1 (7-36) amide (100 nM) was added 4 hours before the cytokines and remained present for the entire cytokine incubation period (24 hours). Apoptosis was assessed by scoring the number of cells expressing GFP and displaying pyknotic nuclei labeled with Hoechst 33342 (10 µg/ml).

Supplemental Figures

Figure S1:GLP-1 increased IRS-2 protein expression in MIN6 cells as assessed by western blot analysis.

Figure S2:Akt phosphorylation in MIN6 cells pretreated or not with GLP-1.

MIN6 cells were treated (+) or not (-) for 18 hours with GLP-1 to increase IGF-1R expression, then incubated with 2 mM glucose for 2 hours, and exposed for 1 hour with 2 or 20 mM glucose. Quantification of Akt phosphorylation is indicated in the lower panel.

Figure S3:Western blot analysis of IGF-1R expression by islets from Gipr-/-;Glp-1R-/- mice infected with recombinant adenoviruses for GFP or IGF-1R expression and treated (+) or not (-) with cytokines. The protein extracts are from the same islets used in Figure 6F.