Collagen 1 coated culture dishes rat tail

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).

Murine keratinocytes from the epidermis of newborn cortactin-deficient (CTTN^−/−) and cortactin wildtype (CTTN^+/+) mice were isolated and immortalized according to the literature for preparation of mouse keratinocytes (19 (link)–21 (link)). In brief, the skin was taken and incubated for 16 h in 2.4 U/ml dispase II in PBS supplemented with Gentamicin/AmphotericinB (CELLnTEC, Bern, Switzerland) at 4°C. After separating the dermis and epidermis, the epidermis was incubated for 20 min with accutase (CELLnTEC) at room temperature, in order to dissociate the cells. Mouse keratinocytes were resuspended and then grown in complete FAD medium (0.05 mM CaCl) on collagen I-coated culture dishes (rat tail; BD Bioscience, New Jersey, USA). The cells were cultivated in a humidified atmosphere containing 5% CO₂ at 35°C. After reaching confluence, cells were switched to 1.2 mM Ca²⁺ and used for experiments after 48 h.