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Blocking of pathogenic anti-Dsg3 antibody-induced increase in keratinocyte stiffness by caspase inhibition, but not Fas ligand neutralization.

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posted on 08.09.2014, 03:10 by Kristina Seiffert-Sinha, Ruiguo Yang, Carmen K. Fung, King W. Lai, Kevin C. Patterson, Aimee S. Payne, Ning Xi, Animesh A. Sinha

HaCaT keratinocytes were grown to confluence and cell stiffness was measured by AFM (expressed as percent of cells measured at a given Young’s Modulus). Cell stiffness distribution in (A) untreated cells (Gaussian fit with peaks around 32.0 kPa and 75.2 kPa), (B) cells treated with the pathogenic anti-Dsg3 antibody Px4-3 (10 µg/ml) alone for 8 h (peaks around 34.8 kPa and 83.5 kPa), (C) cells pretreated with caspase inhibitor (20 µM) 30 min before addition of pathogenic antibody (single peak around 17.7 kPa), (D) cells pretreated with Fas ligand neutralizing antibody (0.5 µg/ml) 30 min before addition of pathogenic antibody (peaks around 29.1 kPa and 73.0 kPa), (E) treated with caspase inhibitor (20 µM) alone for 8.5 h (single peak 30.1 kPa), or (F) treated with Fas ligand neutralizing antibody (0.5 µg/ml) alone for 8.5 h (single peak 32.3 kPa. The single peak of cells of low stiffness observed for treatment with caspase inhibitor (E) and FasL neutralizing antibody (F) alone indicates that the spontaneous apoptosis seen in untreated cells can be completely abrogated by these two blockers.

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