Microarray analysis of RABV-infected neurons isolated by FACS 3 months after infection indicates dysregulation of genes involved in nervous system function and cellular assembly.

<p>Cell suspensions prepared from whole mouse brains 3 months post-infection were sorted on a MoFlo cell sorter for EGFP+ (previously infected) and EGFP- (uninfected) cell populations. The 1248 transcripts differentially expressed between infected and uninfected cells (≥1.5 fold change, p<0.05) were analyzed by Ingenuity Pathway Analysis (IPA) to identify biological functions most significantly affected by the infection (significance predicted by p-value). Shown are the top ten most significant biological systems affected by the gene dysregulation, with the horizontal bars representing the negative log of their p-value (greatest significance at the top). Below each bar is the top three sub-categories affected by gene dysregulation in the respective categories. Each category/sub-category has the number of genes involved (up or down-regulated).</p

EGFP+ neurons are positive for RABV antigen.

<p>Brains were collected from Cre reporter mice fifteen days post-infection, cryosectioned, and EGFP+ regions compared to cell-specific labeling, A) NeuN (blue, neuronal nuclei antibody, 20× fluorescence imaging), B) GFAP (blue, astrocyte antibody, 40× confocal imaging), or C) RABV P antigen (purple) and DAPI nuclear stain (blue, 63× confocal imaging). White arrows in (C) indicate regions positive for RABV P.</p

<i>In vivo</i> analysis of RABV-infected cells using Cre reporter mouse model.

<p>A) Timeline of mouse experiment. Cre reporter mice were infected intranasally (IN) with 10⁵ ffu RABV-Cre and sacrificed at the specified times. B) Weights of infected mice were monitored as a measure of disease throughout the experiment and demonstrate productive infection in all mice within this experiment. C) Brains collected at different time points post-infection were analyzed for the presence of EGFP-expressing cells in the following anatomical regions: olfactory bulb (OB), cerebral cortex (CC), cerebrum (CR), hippocampus (HIP), cerebellum (CB) and midbrain/hindbrain (MB-HB).</p

Dsg2 enhances cystatin A expression <i>in vivo</i>.

<p>(A) Western blot analysis of skin lysates from 3 newborn and 3 adult C57Bl6 mice shows high expression of Csta in newborn but virtually undetectable in adult skin. Actin was used as a control for equal loading. (B) Immunofluorescent staining confirms the Western blotting results showing high level of Csta in newborn wild-type mouse skin. Enlarged image in inset shows cytoplasmic as well as nuclear staining for Csta. (C) Western analysis for Dsg2 and Csta in adult wild-type and Inv-Dsg2 transgenic mouse skin. The results showed expression of the Flag-tagged Dsg2 and Csta in the transgenic but not wild-type mice. Actin showed equal loading. (D) Immunofluorescence was performed on adult skin of wild-type and transgenic mice revealing increased levels CSTA in transgenic skin. Nuclei were counter-stained with DAPI (blue).</p

Differentially expressed genes in response to Dsg2.

<p>Differentially expressed genes in response to Dsg2.</p

Loss of CSTA leads to destabilized intercellular connections.

<p>Cells were treated with non-targeting pool scrRNA or with <i>CSTA</i> siRNA (CSTA KD) followed by mechanical stretching for 4 hr. Cells were allowed to adhere, fixed, and immunostained for Dsg2 (A) and cytokeratin 14 (B) or lysed in Laemmli buffer and immunoblotted for desmoplakin (C). Knockdown of CSTA in keratinocytes resulted in cytoplasmic relocalization of Dsg2, breakage of cytokeratin intercellular connections, and loss of the desmosomal protein, desmoplakin.</p

Top ten genes up- or down-regulated in response to Dsg2.

<p>Top ten genes up- or down-regulated in response to Dsg2.</p

Upregulation of cystatin A, S100 and cyclin genes in Dsg2 transgenic mice.

<p>Upregulation of cystatin A, S100 and cyclin genes in Dsg2 transgenic mice.</p