Figure 8.
The transcriptional activity of the Granuphilin promoter and the cellular content of Granuphilin and Noc2 are linked to the expression level of ICER. (A) INS-1E cells were transiently transfected with plasmids encoding a luciferase reporter gene driven by a minimal promoter (pGL3 basic), by wild-type human Granuphilin promoter (Graluc) or by the human Granuphilin promoter in which the putative CRE was mutated (Graluc mCRE). These plasmids were co-transfected either with an empty vector (empty bars) or with a plasmid leading to the overexpression of ICERIγ (filled bars). Luciferase activity was measured 2 days later. (B) INS-1E cells transiently transfected with an empty vector (−) or with an ICER-Iγ expression plasmid (ICER) were incubated for 2 days at 2 mM glucose, 20 mM glucose or at 2 mM glucose in the presence IBMX/Forskolin (I/F). The expression of Granuphilin, Noc2 and Tomosyn was assessed by Western blotting. (C) INS-1E cells were transiently transfected with the plasmid encoding a luciferase reporter gene under the control of the Granuphilin promoter (Graluc) and with an empty vector or a vector encoding ICER antisense (ICER AS). The cells were culture at 2 mM glucose (G2), 20 mM glucose (G20) or at 2 mM in the presence of Forskolin and IBMX (I/F). The luciferase activity was measured 2 days later. (D) Same conditions as in (C) except that the luciferase reporter gene is driven by a Granuphilin promoter in which the CRE element is mutated (mCRE). (E) INS-1E cells transfected with an empty vector or with ICER antisense (ICER AS) were incubated at 2 mM glucose, 20 mM glucose or at 2 mM glucose in the presence of IBMX and Forskolin (I/F). The expression of Granuphilin and Noc2 was assessed by Western blotting. The results are representative of three independent experiments.