Figure 3

AP-1 mediated gene transcription induced by IGF-II via IR-B. A. HeLa cells transiently transfected with pcDNA3-IR-B or the EV assayed by Western blot detecting the expression of IR and β-actin (loading control). B. Luciferase activity on cells transfected with pAP1-Luc and EV. After 24 hours, serum was depleted for 1 day and cells were stimulated for 16 hours with: 1.6 nM EGF, 100 nM rhIns, 100 nM IGF-II, 1.6 nM EGF + 100nM rhIns or 100 nM IGF-II + 100 nM rhIns. Luciferase induction was calculated as the ratio between stimulated and non-stimulated cells. The results are shown as the mean ± s.e.m (n = 4 independent experiments for EGF, rhIns and EGF + rhIns; n = 3 independent experiments for IGF-II and IGF-II + rhIns and n = 7 for rhIns). Asterisks indicate significant differences (p = 0.005) with non transfected cells (control). C. Cells cotransfected with pAP1-Luc and pcDNA3-IR-B or EV were stimulated with: 1.6 nM EGF, 100 nM rhIns, 100 nM IGF-II, 1.6 nM EGF + 100nM rhIns or 100 nM IGF-II + 100 nM rhIns for 16 hours and luciferase activity was measured. The results are shown as the mean ± s.e.m. (n = 4 independent experiments for EGF, rhIns and EGF + rhIns; n = 3 independent experiments for IGF-II and IGF-II + rhIns). Asterisks indicate significant differences (p < 0.01) between cells over-expressing IR-B with cells transfected with EV. In B and C panels the symbol # indicates that AP-1-Luc response after EGF + rhIns stimulation is significantly different form rhIns alone either in cells over-expressing IR-B (p = 0.007) or in cells transfected with EV (p = 0.005).