Figure 4.

Keratinocytes treated with PV sera and AK23 against Dsg3 shows transient ROS overproduction. Oral keratinocytes OKF4 seeded at approximately 70% confluent density in 96-well plate (i.e. ~1x10⁵ cells per well) in KSFM overnight before treated with PV sera (40% in KGM) (A) (pooled from 2 out of 3 independent experiments) and AK23 (B) (at least 2 experiments) with elevated concentrations or for increasing periods up to 24 hours. Iso Ct: Isotype control IgG1. Cells were then incubated with CellRox reagent (5µM) for 30 minutes before brief washing with PBS followed by image acquisition with an INCA 2200 Analyzer system straightaway. Transient increase of ROS was detected in cells treated with PV sera or AK23 compared to the respective controls, with a dose-dependent response for AK23 (n=25 automated fields/well, mean ± SEM, one-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Of note, a few wells treated with PV sera had a large proportion of cells lost and were excluded from the analysis. The data shown were representative of three independent experiments.