Figure 8.

Inhibition of other kinase signaling pathways can also rescue the H₂O₂ induced YAP accumulation. (A) YAP expression in cells treated with H₂O₂ in the presence and absence of various inhibitors for p38MAPK (SB203580 (SB), 20μM), JNK (SP600125 (SP), 20μM) and PKC (Ro 31-7549 (Ro), 2μM and Go6976 (Go), 1μM), respectively. Cells seeded at ~70% confluent densities (i.e. 2x10⁵ cells per 24-well) were treated with the inhibitors for 1 hour before addition of H₂O₂ and incubated further for 4 hours before fixation and immunostaining for YAP. Both OKF4 and N/TERT cell lines were analyzed by image quantitation for total YAP expression (n=5, representative of 2 experiments, Mean ± SEM, one-way ANOVA was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). The corresponding confocal images of YAP staining are displayed in Figure S4 . Nuc, nucleus; Cyto, cytoplasm. (B) Immunofluorescence analysis of YAP staining in primary skin keratinocytes treated with PV sera (two cases) in the presence and absence of one dose of antioxidants, i.e. GSH (10µM) or NAC (2mM) for 24 hours. Cells seeded in 96-wells at ~70% confluence (i.e. ~1.2x10⁵ cells per 96-well) in KSFM before being exposed to PV sera with -/+ GSH/NAC in calcium-containing KSFM (Ca⁺⁺ 1.5mM) (n=5~7 fields/sample, representative of 3 experiments in three cell lines, Mean ± SEM, *p < 0.05, **p < 0.01). Both GSH and NAC showed to blunt the PV sera-induced YAP levels to some extent compared to PV sera treated cells. (C) Schematic diagram illustrating that PV-IgG targeting Dsg3 causes oxidative stress in keratinocytes with ROS overproduction that, in turn, elicits disruption of cell junction assembly proteins such as Dsg3 and α-catenin, leading to YAP accumulation (dysregulation) and ultimately, pemphigus acantholysis. ROS mediated YAP dysregulation may also be triggered by activations of other signaling pathways such as p38MAPK, JNK and PKC, and inhibition of these pathways can effectively prevent YAP accumulation. The green dotted bar-headed line indicates the negative feedback mechanism of YAP regulation to Dsg3.