Fig. 6. Altered p53 expression and distribution were detected in keratinocyte cultures treated with PV sera and pathogenic antibody.

a Confocal microscopy of NTERTs treated with the PV sera, dual labeled for Dsg3 and p53. Cells were seeded at confluent densities in KGM for overnight before being treated with PV sera (at 40% concentration in KGM) from a different cohort of PV patients (n = 17), for 24 h. Disruption or depletion of Dsg3 at the plasma membrane accompanied with marked increases in p53 was observed in PV serum-treated cells that were abolished by p53 knockdown, compared to controls exposed to sera of healthy individuals that displayed, predominantly, nuclear p53 signals. Of note, p53 also showed distribution at the membrane where it colocalized with the fragmented Dsg3 (arrows in the inserts). Additional data for p53 knockdown was shown in Supplementary material Fig. S5. The image magnification in PV serum-2 was doubled, relative to other panels, to highlight the disruption of junctions and peripheral distribution of p53. b Scatter and whisker plots of Dsg3 and p53 cellular and subcellular expression (n = 16 for PV serum samples, n = 6 for control samples). Student t-test and the Wilcoxon–Mann–Whitney Rank Test were used for statistically significant analysis here and gave similar results. c The relative ratio of p53 nuclear versus cytoplasmic cellular distribution in controls and 16 PV sera treated NTERTs. d Treatment of NTERTs with the pathogenic monoclonal antibody AK23 targeting Dsg3 N-terminus, caused p53 induction, in a time and dose-dependent manner (n = 11 fields per condition, pooled from 2 independent experiments). For the dose-response experiment, cells were treated with AK23 for 6 h. e Confocal images of cells with triple staining, treated in the presence and absence of AK23. Disruption of F-actin along with Dsg3 (arrowheads) was readily detectable in cells exposed to AK23. Increase p53 expression was detected predominantly in the nucleus and also was observed at the plasma membrane where it showed colocalization with Dsg3 and F-actin (arrows) in cells treated with AK23. The membrane distribution of p53 was not detectable in control cells. The protein colocalization of the dotted line box is shown at the bottom. (*p < 0.05, **p < 0.01, ***p < 0.001). Scale bar, 10 µm. f Schematic model of Dsg3 in the suppression of p53 in keratinocyte response to stresses. Disruption of Dsg3 by RNAi or PV autoantibodies evokes p53 induction