A. Induction of apoptotic processes by pathogenic anti-Dsg3 antibodies.
Confluent HaCaT cell cultures were treated with 10 µg/ml of either the pathogenic anti-Dsg3 antibody Px4-3 or the non-pathogenic anti-Dsg3 antibody Px4-4 for 4, 8, 12, 16, 20 and 24 h. Induction of apoptosis was assessed by measuring the expression of both Annexin V and PI positivity by FACS. Each experiment was performed in triplicate. Values are presented as percent induction when compared to baseline apoptosis levels in untreated cells harvested in parallel. The levels of annexin V and/or PI positivity induced by pathogenic anti-Dsg3 antibodies are significantly elevated at 12, 16 and 20 h when compared to untreated cells (p≤0.05, Student’s t-test). B and C. Fas ligand-induced apoptosis correlates with a marked increase in keratinocyte stiffness. Subconfluent HaCaT cell cultures were left untreated (B1), treated with 50 ng/ml FasL (B2) or pretreated with FasL neutralizing antibody (0.5 µg/ml) for 30 min before addition of 50 ng/ml FasL (B3). In untreated HaCaT cells, 4.52±0.32% of all cells are Annexin V- and PI positive as measured by flow cytometry after 44 h of culture. Addition of FasL significantly increases the number of apoptotic cells to 14.63±0.11% (p<0.01, FasL vs. untreated). Pretreatment of FasL-treated cultures with FasL neutralizing antibody significantly reduces FasL-induced apoptosis back to baseline levels 5.95±1.28% (p<0.01, FasL + FasL inhibitor vs. FasL alone). In parallel experiments, 100 cells were randomly selected to collect AFM force-displacement curves to generate a Young's modulus value for each cell. The combined Young’s modulus values for each condition were plotted as Gaussian distribution curves. (C1) Normal cell stiffness distribution in untreated cells (Gaussian fit with peaks around 31.2 kPa and 77.8 kPa). (C2) Cell stiffness distribution in Fas ligand-treated cells (Gaussian fit with peaks around 31.5 kPa and 81.7 kPa). (C3) Cell stiffness distribution in Fas ligand treated cells pretreated with Fas ligand neutralizer (Gaussian fit with single peak around 29.1 kPa). Data are representative of three independent experiments performed in duplicate.