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. 2016 Feb 18;7(2):e2107. doi: 10.1038/cddis.2016.21

Figure 1.

Figure 1

(a) INS-1E cells were transfected with either control Scr siRNA (siScr) or NF2 siRNA (siNF2) (both from Dharmacon, Lafayette, CO, USA) and treated with 22.2 mM glucose or the mixture of 22.2 mM glucose and 0.5 mM palmitate (22.2  Palm) for 2 days. Human islets were infected with either control Ad-GFP-Scr or Ad-GFP-hShNF2 adenoviruses (VectorBioLabs, Malvern, PA, USA) for 1 day and then treated with the mixture of 22.2 mM glucose and 0.5 mM palmitate or the pro-inflammatory cytokines 2 ng/ml recombinant human IL-1β and 1000  U/ml IFNγ (IL/IF) for 3 more days. INS-1E cells were transfected with either control siScr or siNF2 and treated with 22.2 mM glucose for 2 days. INS-1E cells were transfected with either control siScr or siNF2 for 1 day and then infected with either Ad-LacZ control or Ad-LATS2 adenoviruses (VectorBiolab) and then treated with 22.2 mM glucose for another day. (b) INS-1E cells and isolated human islets were transfected with either control siScr or siNF2. Cleaved capase-3, cleaved PARP, NF2, pMST1, pLATS1/2, LATS2, pS6K and p4EBP1 were analyzed by western blotting. Western blots show representative results from three independent experiments (INS-1E) from three different donors (human islets). Tubulin was used as loading control. (c) Human islets were infected with either control Ad-GFP-Scr or Ad-GFP-hshNF2 adenoviruses (VectorBiolab) for 2 days. Insulin secretion during 1 h incubation with 2.8 mM (basal) and 16.7 mM glucose (stimulated), normalized to insulin content. The insulin stimulatory index denotes the ratio of secreted insulin during 1 h incubation with 16.7 and 2.8 mM glucose. Pooled data from three independent experiments from three different donors (human islets). Quantitative RT-PCR for NF2, Insulin, Pdx1, NeuroD1, Slc2a2, MafA, GCK, Nkx6.1, Nkx2.2, GCG and Glis3. For analysis, we used the AppliedBiosystems StepOne Real-Time PCR system (Applied Biosystems, Carlsbad, CA, USA) with TaqMan(R) Fast Universal PCR Master Mix for TaqMan assays (AppliedBiosystems). Pooled data from four independent experiments from four different donors (human islets)