Journal: Frontiers in Immunology

Article Title: Oxidative Stress-Mediated YAP Dysregulation Contributes to the Pathogenesis of Pemphigus Vulgaris

doi: 10.3389/fimmu.2021.649502

Figure Lengend Snippet: Expression of exogenous α-catenin or treatment of keratinocytes with antioxidants can rescue H 2 O 2 inducted YAP nuclear accumulation with concomitant enhancement of the cell junctional integrity. (A) Analysis of YAP expression and distribution in GFP+ (indicating α-catenin-GFP transfection) and GFP- cells (α-catenin-GFP un-transfected cells), respectively. Cells transfected with empty vector served as the control. Expression of α-catenin-GFP caused a reduction of nuclear YAP. (B) Confocal images of α-catenin-GFP plasmid transfected or mock control N/TERTs seeded at 2.5x10 5 per well to reach ~70% confluent densities. Next day cells were treated with or without H 2 O 2 (only one dosage was displayed) for 2 hours and doubled labeled for YAP (green) and GFP (red). YAP nuclear exclusion was evident in cells with α-catenin-GFP expression. (C) Image quantitation in different conditions shown in B (n=5 fields/coverslip, representative of 2 independent experiments, Mean ± SEM, normalized to PBS control nuclear signal that was arbitrarily set as 1). The green line indicates comparison of total fluorescent intensity between control and α-catenin-GFP transfected cells, whereas the black lines were comparisons for cytoplasmic (Cyto) and nuclear (Nuc) signals, respectively. (D) YAP expression in cells of ~70% confluence, treated with one dosage of H 2 O 2 in the presence and absence of GSH (10μM) and NAC (2mM) for 2 hours (n=13, pooled data from 3 independent attempts, Mean ± SEM). Cells were treated with antioxidants 1 hour before H 2 O 2 addition. Confocal images for YAP and Dsg3 (inserts, stained with 5H10 mAb) were displayed underneath. (E) Dispase assay in different conditions as shown in D. Cells of 100% confluence grown in KGM were treated with H 2 O 2 in the presence and absence of GSH (10μM) and NAC (2 mM) for 4 hours (n=5, pooled from 3 independent experiments, with one in triplicate, Mean ± SD),. One-way ANOVA was used to obtain all p values. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, and NS indicates no significance. Scale bars, 10µm.

Article Snippet: Briefly, 2x10 5 cells were seeded into a 6-well plate overnight before transfected with α-catenin-GFP (gift from Ann Wheeler) or pEGFP-C3-hYAP1 (Addgene Plasmid #17843) or pENTR1A-Yap S127A (Addgene, Plasmid #46050) expression vector along with an empty vector plasmid (pBABE-puro served as the control) using FuGENE HD transfection reagent (E2311, Promega).

Techniques: Expressing, Transfection, Plasmid Preparation, Control, Labeling, Quantitation Assay, Comparison, Staining

Journal: Frontiers in Immunology

Article Title: Oxidative Stress-Mediated YAP Dysregulation Contributes to the Pathogenesis of Pemphigus Vulgaris

doi: 10.3389/fimmu.2021.649502

Figure Lengend Snippet: Expression of exogenous YAP causes disruption in the junctional assembly of α-catenin and Dsg3. (A) Confocal images of N/TERTs transfected with GFP-YAP or empty vector plasmid (Vect Ct), double-stained for GFP (green) and α-catenin (red). Cells were seeded at 2.5x10 5 per well on coverslips in a 24-well plate in KSFM to reach ~70% confluence. The next day, the mediums were changed to KGM and cells were incubated for various periods before fixation (2 experiments). Inserts showed enlarged fields of the square boxes with GFP positive cells in which disruption of α-catenin at cell junctions was evident. (B) Image quantitation for α-catenin staining in cells transfected with YAP-S127A plasmid or Vect Ct and cells were treated in the same way as described in (A) . A significant reduction in α-catenin expression was detected at 2 hours and 20 hours, especially at the later time point, compared to controls (n=5 fields/sample, 2 experiments, **p < 0.01). (C) Image quantitation for Dsg3 (with 5H10) and YAP staining in a parallel set of coverslips that indicated increased nuclear YAP in cells transfected with YAP-S127A compared to Vect Ct cells (n=5 fields/sample, 2 experiments, *p < 0.05). A reduction of Dsg3 was also detected at 20 hours’ time point in YAP-S127A transfected cells compared to control (*p < 0.05). (D) YAP luciferase assay indicated a trend of increased luciferase activities in both GFP-YAP and YAP-S127A transfected cells compared to Vect Ct. (E) Western blotting for the indicated proteins in N/TERTs transfected with either scrambled (Scram) or siRNA-1/2. Increased expression of cell adhesion proteins was shown in YAP knockdown cells compared to controls (at least 3 experiments, unpublished data in other cell lines). (F) Image quantitation of Dsg3 staining in N/TERTs transfected with scrambled (Scram) or siRNA-1/2 and treated with isotype IgG control (Iso Ct) and AK23 (2µg/ml), respectively. 1x10 5 siRNA transfected cells were seeded in 96-well in KSFM overnight before IgG treatment in calcium-containing (1.2mM) KSFM for 24 hours. It showed YAP depletion resulted in a 2~3-fold increase of Dsg3 expression compared to Scram control cells treated with Iso Ct IgG, which blunted the effect of AK23 induced Dsg3 reduction (n=8~9 fields/sample, *p < 0.05, ****p < 0.0001). Scale bars, 20µm.

Article Snippet: Briefly, 2x10 5 cells were seeded into a 6-well plate overnight before transfected with α-catenin-GFP (gift from Ann Wheeler) or pEGFP-C3-hYAP1 (Addgene Plasmid #17843) or pENTR1A-Yap S127A (Addgene, Plasmid #46050) expression vector along with an empty vector plasmid (pBABE-puro served as the control) using FuGENE HD transfection reagent (E2311, Promega).

Techniques: Expressing, Disruption, Transfection, Plasmid Preparation, Staining, Incubation, Quantitation Assay, Control, Luciferase, Western Blot, Knockdown