Differential gene expression between Inv-Dsg2 transgenic and wild-type mouse skin.

<p>(A) Total RNA was isolated from the skin of 2 wild-type and 2 Inv-Dsg2 transgenic mice, reverse transcribed, biotin-labeled and applied to a mouse cDNA microarray. The dendogram (heat map) shows that 492 genes were either up-regulated (red/orange) or down-regulated (blue/green) in transgenic (T1 and T2) and control (W1 and W2) mice. (B) Volcano plot shows the log2 (fold change) in x-axis versus the—log10 (p value) in the y-axis. The points having a fold-change less than 2 (log2 = 1) are shown in gray. The vertical green lines demarcate where the fold change equals 2 (right line) or equals—2 (left line). The horizontal green line demarcates where the p value is 0.05, with points above the line having p<0.05 and points below the line having p>0.05. Depicted in red are the genes that exhibit a greater than 2 fold change with a p>0.05 in transgenic epidermis as compared to control. The arrows indicate genes of interest. (C) Quantitative real-time RT-PCR analysis reveals an average of 34.33±1.32 fold increase in Dsg2 RNA expression in Inv-Dsg2 transgenic (Tg) compared to that of wild-type (WT). In addition, RNA expression for transgenic relative to control were: <i>Csta1</i>, 113.24 ±2.23; <i>Csta2</i>, 1227.04 ±1.26; <i>Csta2l1</i>, 1.11 ±0.47; <i>Csta3</i>, 26.97±0.44 (Bar = mean ± s.d.; (*p< 0.05; **p<0.01; ***p<0.001; Student’s <i>t</i> test).</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

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

Modulation of cell adhesion by CSTA and Dsg2.

<p>(A) Immunofluorescence of normal human skin (A) and palm (B) showing low levels of Dsg2 in the basal layer (arrows) of the normal skin (inset: enlarged image) but high levels in both the basal and differentiated layers (arrow head) in the palm. Note: Immunostaining was performed at the same time and images were captured at the same exposure. (B) A431-shGFP and A431-shDsg2 cells were treated with scrambled RNA or siRNA to <i>CSTA</i> for 72 hr and then subjected to the <i>in vitro</i> mechanical stress dispase-based dissociation assay. Bright field images showing loss of Dsg2 or CSTA induced fragmentation and the loss of both had a synergistic effect on cell adhesion. (C) Graph showing the number of fragments for each condition. i, shGFP; ii, shGFP + scrRNA; iii, shGFP + siCSTA; iv, shDsg2; v, shDsg2 + scrRNA; vi, shDsg2 + siCSTA. Bar = mean ± s.e.m. *p< 0.05 and **p<0.01 using Student’s <i>t</i> test.</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