Different effect of IFN-γ on IDO expression in dermal fibroblasts of C57BL/6 prediabetic NOD mice.

<p>Dermal fibroblasts from prediabetic (8 weeks of age) male and female NOD mice failed to respond to IFN-γ induced IDO. Dermal fibroblasts isolated from C57BL/6 male mice of 8 weeks of age as control (solid bars), and aged matched male (hatched bars) or female (open bars) prediabetic NOD mice were treated with 1000 U/ml of IFN-γ for 48 hours. <b>A</b>: Kyn levels in CM of treated cells, <b>B</b>: IDO expression at the protein level, <b>C</b>: the Mean±SEM ratio of densities of IDO to β-actin at protein control group treated with IFN-γ (n = 3, p<0.01). β-actin expression showed equal loading of proteins. ND: not detected.</p

COL-I expression in dermal fibroblasts from control and NOD mice.

<p>COL-I expression in dermal fibroblasts from C57BL/6 (solid bars) and NOD (open bars) mice was evaluated by western blot and RT-PCR analyses. Cells were exposed to 0 or 1000 U/ml of IFN-γ for 48 hours before analysis. <b>A</b>: COL-1 expression at the protein level. <b>C</b>: COL-1 expression at mRNA level. <b>B</b> and <b>D</b> represent the Mean±SEM ratio of COL-1 to β-actin at protein and mRNA levels respectively. β-actin was used as loading control in both western blotting and RT-PCR assays. *demonstrates significant difference between C57BL/6 and NOD fibroblasts treated with IFN-γ in terms of COL-1 expression. **corresponds to significant difference between cells from the same strain treated with 0 or 1000 U/ml of IFN-γ (n = 3, p<0.05).</p

IDO protein and mRNA expression in Ad-IDO transfected cells.

<p>Dermal fibroblasts from C57BL/6 (solid bars) and NOD (open bars) mice were transduced with Ad-IDO or mock vector. <b>A</b>: IDO expression was analyzed by western blotting, <b>C</b>: IDO expression was analyzed by RT-PCR. <b>B</b> and <b>D</b>: the Mean±SEM ratio of IDO to β-actin at the protein and GAPDH at mRNA level (n = 3). β-actin and GAPDH were used as a loading control for protein and mRNA expression respectively. ND: not detected.</p

MHC-I mRNA expression in fibroblasts isolated from control and NOD mice.

<p>Cells were treated with 0 or 1000 U/ml of IFN-γ for 48 hours. <b>A</b>: RT-PCR analysis of MHC-I mRNA expression. <b>B</b>: the Mean±SEM ratio of densities of MHC-I to GAPDH. Solid and open bars represent C57BL/6 and NOD fibroblasts respectively. GAPDH was used as loading control. *denotes significant difference between C57BL/6 and NOD fibroblasts treated with IFN-γ in terms of MHC-I expression (n = 3, p<0.05). **corresponds to significant difference between cells from the same strain treated with 0 or 1000 U/ml of IFN-γ (n = 3, p<0.01).</p

IFN-γ-induced-STAT1 phosphorylation in C57BL/6 and NOD dermal fibroblasts.

<p>Following starvation for 18 hours, dermal fibroblasts from NOD (open bars) and C57BL/6 (solid bars) mice were remained untreated or treated with 1000 U IFN-γ per ml of DMEM plus 2% FBS for 15, 30 or 60 minutes. Cell lysates were collected for western blot analysis. <b>A</b>: STAT 1 phosphorylation shown by western blotting. <b>B</b>: the Mean±SEM ratio of phospho-STAT1 (P-STAT1), to the ratio of β-actin to total STAT1. Total STAT1 and β-actin expressions were used as loading controls. *denotes significant difference between related bars (p<0.05, n = 3). UT: untreated, ND: not detected.</p