Influence of Ocln on epidermal differentiation and EMT markers, cell-cell adhesion and paracellular Ca²⁺ permeability.

(A) (left side) Western Blot analysis of 3D skin models after silencing of Ocln with 2 different siRNAs shows downregulation of Ocln and of the epidermal differentiation marker involucrin and an upregulation of TG1. There is no alteration for the EMT markers E-cadherin and vimentin. Same amounts of protein were loaded and actin or tubulin were used as gel loading controls. A representative experiment is shown (n = 3). (right side) Semiquantitative analysis of Ocln, involucrin, TG1, E-cadherin and vimentin. Band intensities were normalized to actin (Inv, TG1) or tubulin (E-cad, vim). Subsequently, the values were normalized to control siRNA treated cells (n = 3; mean±SEM; *p<0.05, ***p<0.001 compared to control siRNA). (B) Calcium induced cell-cell adhesion was investigated with a hanging drop assay at the indicated time points. Significantly greater numbers of particles (indicating less cell-cell adhesion) were found in the suspensions of Ocln knock-down cells compared to the controls (*: p<0.05 n = 3), (C) Electrophysiological studies of paracellular Ca²⁺-permeability in Ocln siRNA-treated cultured keratinocytes revealed an increase in paracellular permeability for Ca²⁺ in Ocln knock-down cells compared to cells treated with control siRNA (n = 6, mean ± SEM). (D) Example for the knock-down of Ocln in siRNA treated submerged cells. Mean knock-down of Ocln in the cells used for experiments in Figure 5 B, C and 6 was 76% +/−9%.