Deregulation of c-Fos expression induces phenotypic and functional defects in DCs. (A) Expression of c-Fos protein was analyzed by Western blotting in untransduced DC²¹¹⁴ cells and DC²¹¹⁴ cells transduced with an empty expression vector or a c-Fos expression vector. Actin was used as internal control. A representative gel is shown. c-Fos signals were quantified and normalized relative to actin. Changes (-fold) are indicated above the gel. (B) c-Fos mRNA was quantified by real-time RT-PCR in CpG-treated DC²¹¹⁴ cells transduced with an empty expression vector or a c-Fos expression vector. A representative graph of 2 independent experiments is shown. (C) Unstimulated and CpG-treated DC²¹¹⁴ cells transduced with an empty expression vector or a c-Fos expression vector were examined by immunofluoresence microscopy, and frequencies of cells exhibiting a characteristic dendritic morphology were determined. Representative images are shown at the left; dendritic protrusions are indicated with arrows. The bar graph represents the means and SDs derived from 3 independent transductions; ns, not significant; *P < .05. (D) Cell-surface CD86 and CD40 expression was analyzed by flow cytometry for unstimulated and CpG-stimulated DC²¹¹⁴ cells. The histograms (top) are representative of at least 3 experiments. The graphs (bottom) represent the mean fluorescence intensities (MFI) for cell-surface CD86 and CD40 expression, and show the means and SDs derived from at least 3 independent experiments; *P < .05. (E) CpG-treated DC²¹¹⁴ cells transduced with an empty vector or a c-Fos expression vector were loaded with OVA peptide (left panels) or OVA protein (right panels) and cocultured with OVA-specific CD4⁺ T cells purified from TCR-transgenic OTII mice. DC²¹¹⁴ cells that had not been loaded with antigen (−OVA) were used as negative controls. T-cell activation was determined by the analysis of cell-surface CD69 expression (top panels, relative frequencies of CD69⁺ cells) or secretion of IL2 into the supernatants (bottom panels, relative IL2 secretion). The means and SDs derived from 3 independent experiments are shown; *P < .05; **P < .01; ***P < .001.