Protocol detail

Generation of Vinculin and E-cadherin Deficient Keratinocytes

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Vinculin and E-cadherin-deficient cells were generated from epidermal, murine keratinocytes as described before (Michels et al., 2009 (link); Mertz et al., 2013 (link); Rubsam et al., 2017 (link)). All cells were cultivated under sterile conditions at 32°C and 5% (vol/vol) CO₂ in low calcium (<0.5 mM) containing DMEM/Ham´s F12 medium (Biochrom, Berlin, Germany) supplemented with 10% FCS Gold (chelex treated) (PAA laboratories, Cölbe, Germany), 1% glutamine (Biochrom, Berlin, Germany), 1% penicillin/streptomycin (Biochrom, Berlin, Germany), 0.18 mM adenin (Sigma, St. Louis, MO), 0.5 µg/ml hydrocortisone (Sigma), 5 µg/ml insulin (Sigma), 10 ng/ml EGF (Sigma), 0.05 µg/µl choleratoxin (Sigma), and 5 µg/ml vitamin C (Sigma).

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Noethel B., Ramms L., Dreissen G., Hoffmann M., Springer R., Rübsam M., Ziegler W.H., Niessen C.M., Merkel R, & Hoffmann B. (2018). Transition of responsive mechanosensitive elements from focal adhesions to adherens junctions on epithelial differentiation. Molecular Biology of the Cell, 29(19), 2317-2325.

Publication 2018

DMEM/Ham´s F12 medium glutamine penicillin/streptomycin adenin hydrocortisone insulin EGF choleratoxin vitamin C

Adenin Calcium Cells Chelex Choleratoxin E cadherin Epidermal Glutamine Gold Hydrocortisone Insulin Keratinocytes Murine Penicillin Sterile Streptomycin Vinculin Vitamin c

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Variable analysis

independent variables

Vinculin-deficient cells
E-cadherin-deficient cells

dependent variables

Not explicitly mentioned

control variables

Cultivation conditions (32°C, 5% CO2, low calcium (< 0.5 mM) DMEM/Ham's F12 medium)
Supplemented media (10% FCS Gold, 1% glutamine, 1% penicillin/streptomycin, 0.18 mM adenin, 0.5 µg/ml hydrocortisone, 5 µg/ml insulin, 10 ng/ml EGF, 0.05 µg/µl choleratoxin, 5 µg/ml vitamin C)

controls

No positive or negative controls were explicitly mentioned in the provided information.

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