Murine Keratinocyte Differentiation Dynamics

In this study, murine keratinocytes (KtyII) isolated from wild-type (KtyII wt) and keratin cluster II knockout (KtyII k.o.) were used. Cells were immortalized as described elsewhere in detail (22 (link)). Cells were grown in complete FAD media (0.05 mM CaCl₂) on collagen I-coated culture dishes (rat tail; BD). For all experiments, cells were grown to confluency before switching them to high Ca²⁺ (1.2 mM) for 48 h to induce proper differentiation and usage for experiments. For fluorescence recovery after photobleaching (FRAP) experiments, cells were transient transfected at 70% confluency with pEGFP-C1-Dsg3 (kindly provided by Dr. Yasushi Hanakawa, Ehime University School of Medicine, Japan) using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to manufacturers’ protocol. 24 h after transfection, cells were switched to high Ca²⁺ (1.2 mM) and grown for further 48 h before the experiments. Activity of p38MAPK was modulated using either p38 inhibitors SB202190 (Merck, Darmstadt, Germany) and SB203580 (Sigma Aldrich, Munich, Germany) (both 30 µM) or p38 activator anisomycin (60 µM) (Sigma Aldrich, Munich, Germany).