IGF-1R protein differentially accumulates in the nuclei of TAO orbital fibroblasts and derives from the fibroblast surface.

<p>(<b>A</b>) Western blot analysis of nuclear and cytoplasmic IGF-1Rα in GD orbital fibroblasts before and following IGF-1 (10 nM) treatment for 16 h. Cells were subjected to subcellular fractionation as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034173#s4" target="_blank">Methods</a>” and membranes were probed with anti-IGF-1Rα, stripped, and re-probed with anti-Grb2 (cytoplasmic) and anti-c-Jun (nuclear) Abs. (<b>B</b>) Nuclear IGF-1Rα content in GD and control orbital fibroblasts before or following treatment with either IGF-1 (10 nM) or GD-IgG (15 µg/ml) for 16 hours. (<b>C</b>) Insulin fails to alter the nuclear content of IR or IGF-1Rα in GD orbital fibroblasts. Cells were treated with nothing or insulin (15 µg/ml) for 16 hrs. They were subjected to subcellular fractionation and Western blot analysis. (<b>D</b>) IGF-1Rβ (98 kDa) and the intact receptor (200 kDa) are undetectable in the nucleus under basal and IGF-1-treated conditions. (<b>E</b>) Control and GD fibroblasts were subjected to ¹²⁵I-IGF-1 cross-linking with either the cell-impermeable agent, BS, or the cell permeable agent, DSS. They were then treated with IGF-1. Nuclei were separated as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034173#s4" target="_blank">Methods</a>” and subjected to quantification of radioactivity. Results are representative of three experiments performed.</p

siRNA directed against ADAM17 attenuates the nuclear accumulation of IGF-1Rα in GD orbital fibroblasts.

<p>Fibroblasts were transfected with control siRNA or that specifically targeting ADAM 17. Some were left untreated while others were treated with IGF-1. Cells were fixed and stained with anti-IGF-1Rα Ab/Orgeon Green anti-rabbit Ab. Quantification of nuclear intensity was conducted so that each column represents the mean of ten randomly chosen nuclei ± SD. *, p<0.002 vs control; **, p<0.01 vs IGF-1. n = 3 independent determinations. (Inset) Adam17 siRNA or control siRNA was transfected into GD fibroblasts, cells were lysed, and proteins subjected to Western blot analysis by probing with anti-ADAM 17 Ab, stripping the membrane and incubating with anti-β-actin.</p

Confocal imaging of IGF-1R reveals nuclear accumulation in TAO fibroblasts following treatment with either IGF-1 or GD-IgG.

<p>(<b>A</b>) untreated (control, upper panels) or IGF-1 (10 nM) (lower panels) for 16 h. reveals localization of IGF-1Rα (green) as indicated by the yellow overlay nuclei in GD fibroblasts. Chromatin was stained by PI (red). Control fibroblasts failed to respond. The images labeled IGF-1Rβ (green) demonstrate an absence of effects with IGF-1 on nuclear accumulation. Scale bar, 25 µm. (<b>B</b>) IGF-1Rα (green) co-localizes in the PI-stained nuclei (yellow overlay) following treatment with GD-IgG (15 µg/ml) in GD but not in control fibroblasts. (<b>C</b>) Dexamethasone (10 nM) attenuates the nuclear accumulation of IGF-1Rα (<b>D</b>) as does the IGF-1R-blocking mAb, 1H7 (5 µg/ml). These experiments have been performed 4 times.</p

Virally encoded IGF-1Rα-GFP fusion protein expression in GD and control orbital fibroblasts.

<p>(<b>A</b>) Expression of the adenovirus-encoded IGF-1Rα–GFP fusion protein can be detected by Western blot analysis probed by both anti-GFP and anti-IGF-1Rα. (<b>B</b>) IGF-1Rα-GFP fusion protein accumulates in the nuclei of both control and GD orbital fibroblasts, as assessed by Western blot analysis following subcellular fractionation of nuclear protein. IGF-1 treatment fails to alter this pattern. These studies are representative of 5 experiments involving expression of virally encoded IGF-1Rα.</p