FIG. 4.

Ad-mediated overexpression of PHIP1 promotes proliferation and potentiates IGF-1-stimulated mitogenesis of β cells. (A) Immunoblot analysis of PHIP1 expression in INS-1 cells infected with increasing doses of AdGFP and AdPHIP1 (MOI, 100 to 400, as indicated) indicates dose-dependent increase of exogenous PHIP1 expression (left). The IRS2 immunoblot demonstrates the level of IRS2 overexpression in AdIRS2-infected cells (right). Actin expression was used as a loading control. (B) Time course of INS-1 and NIH 3T3 proliferation assessed by MTS assay and counting of live cells, respectively, 24 to 96 h postinfection with AdGFP, AdPHIP1, and AdIRS2 (MOI, 200). Cells were incubated in the presence of 11 mM glucose-10% FBS. Experiments were performed three times in sextuplicate. Results are shown as means ± SD. *, P < 0.05 versus results for AdGFP-infected cells. OD450, optical density at 450 nm. (C) Ad-mediated overexpression of PHIP1 enhances IGF-1-dependent mitogenesis and promotes increase of cyclin D2 protein levels in INS-1 cells. Cells were infected with AdGFP, AdPHIP1, and AdIRS2 (MOI, 200) and, 16 h postinfection, were made quiescent for 24 h. Subsequently, cells were incubated in RPMI medium with 15 mM glucose in the presence (+) or absence (−) of 10 nM IGF-1. BrdU incorporation was measured as indicated in Materials and Methods. The data are expressed as the increases ± standard errors of the means compared to results for control cells (AdGFP-infected cells treated with 15 mM glucose in the absence of IGF-1 [GFP/−IGF group]). Experiments were performed three times in quadruplicate. *, P < 0.05. Bottom panel, cells were treated as described above and lysates were immunoblotted with cyclin D2 antibodies. Actin was used as a loading control. (D) Densitometry analysis from the three independent experiments described for panel C is summarized as a histogram. The data are expressed as the increases ± standard errors of the means. *, P < 0.05 versus results for AdGFP-infected cells treated in the absence of IGF-1; #, P < 0.05 versus results for AdGFP-infected cells treated in the presence of IGF-1. (E) Ad-mediated overexpression of PHIP1 enhances IGF-1-dependent cyclin D2 promoter activity in INS-1 cells. Cyclin D2 luciferase reporter was transfected into INS-1 cells. Subsequently, cells were infected with increasing MOIs, as indicated, of AdGFP or AdPHIP1 and incubated in the presence (+) or absence (−) of IGF-1 (10 nM). Luciferase assay was performed as indicated in Materials and Methods. Experiments were performed three times in triplicate. The data are expressed as the increases ± SD compared to results for control cells (AdGFP-infected cells treated with 15 mM glucose in the absence of IGF-1 [AdGFP/−IGF group]). *, P < 0.05 versus results for AdGFP group infected with same MOI in the absence of IGF-1; #, P < 0.05 versus results for AdPHIP1 group infected with same MOI in the presence of 10 nM of IGF-1. +, present; −, absent.