Figure 2.

Treatment of keratinocytes with PV sera or the pathogenic monoclonal antibody against Dsg3 results in increased YAP expression. (A) Confocal images of N/TERT cells double-labeled for Dsg3 (5H10, red) and YAP (green). Cells were seeded on coverslips at approximately 80% confluent densities (~2.5x10⁵/well) one day before being treated with PV sera (40%) in KSFM (Ca⁺⁺ 90µM) alongside control serum overnight. Then, the medium was replaced with KSFM with normal calcium (1.8mM) plus PV sera at the same concentration of 40% and cultured for further 5 hours (24 hours PV sera in total) before fixation. Coverslips were immunostained for the indicated proteins. Increased cytoplasmic YAP (arrows) with aggregations at the cell borders (arrowheads) were shown in PV serum treated cells coupled with Dsg3 reduction. (B) Image quantitation of YAP staining shown in (A) (n=10 PV patients’ sera, 2 experiments, the comparison was made by two-tailed Student t-test, *p<0.05). (C) N/TERT cells of ~80% confluence grown for one day were treated with AK23, the pathogenic monospecific antibody against Dsg3, in KGM for 6 hours and dual labeled for Dsg3 (5H10) and YAP. The insert showed the characteristic linear arrays of Dsg3 staining (red) at the cell border, the feature of anti-Dsg3 mediated disruption as described previously (42). Elevated YAP levels were shown in both the nuclei (Nuc) and cytoplasm (Cyto) of AK23 treated cells compared to the counterpart treated with isotype IgG1 control (Iso Ct IgG). Reduction of Dsg3 staining was observed in cells treated with AK23. (D) Image quantification for the dose and time-course experiments (normalized to control nuclear signal that was arbitrarily set as 1), with AK23 (2 µg/ml used in the time course study) (n=4 fields/sample, 2 experiments for dose and 3 experiments for time course, mean ± SEM, one-way ANOVA was used to obtain the p values (*p<0.05, ***p<0.001). Data were representative of at least three independent experiments. Scale bars, 10µm.