MKCs were prepared from newborn mice and were cultured following standard protocols, as previously described [37 (
link), 38 (
link)]. In order to obtain CnB1 KO MKCs, MKCs from CnB1
flox/flox transgenic mice (see below) were infected with an adenovirus expressing Cre (Ad-Cre) or GFP (Ad-GFP, as a control). Primary HKCs were isolated from foreskin tissues and were cultured in serum-free keratinocyte medium (SFM, Invitrogen) as previously described [39 (
link)–41 (
link)]. All SCC lines (SCC12, 13, 15, 25 were derived from the skin, and SCCO11, O12, O22, O23, O28 were derived from the oral epithelium) were cultured in SFM. Human foreskin tissues were obtained from discarded hospital specimens following an institutional protocol (NO.2015120401, Date: 12-05-2015). Clinical SCC samples were obtained from the Department of Dermatology, Zurich University Hospital, Zurich, Switzerland. Participants were provided verbal and written informed consent; the protocol was approved by the Swiss Ethics Committee as described previously [26 (
link), 42 (
link)].
Methods for infecting adenoviruses, lentivirus or retroviruses followed described protocols [26 (
link), 43 (
link)]. For transient transfection of siRNA, Lipofectamine 2000 (Invitrogen) was employed and the final concentration of siRNA in the transfection medium was 200 nM. All siRNA oligo sequences are listed in
S2 Table.
CsA (30024, Sigma-Aldrich) was dissolved in DMSO and stored as a stock solution (20 mM); TPA (P8139, Sigma-Aldrich) was dissolved in acetone and stored as a stock solution (10 mM). Both CsA and TPA were kept at -80°C. The Vivit peptide, an inhibitor of calcineurin mediated NFAT activation [44 (
link)], and its negative control, the Veet peptide, were chemically synthesized by the Peptide Core facility of the University of Lausanne and were then dissolved in H
2O and stored as stock solutions (20 mM) at -80°C. The final concentration of CsA and Vivit used to treat in vitro keratinocyte cultures was 5 μm.
For UVB treatment
In vitro: After removal of culture medium and two washes with PBS, confluent keratinocytes were covered with PBS and exposed to 35 mJ/cm
2 UVB. The UVB dose was measured each time using an IL 1400A photometer (International Light Inc., Newburyport, MA) equipped with a SEL240 probe. After UVB exposure, PBS was removed from the cells and replaced with culture medium. Cells were harvested at different times (as indicated in each experiment) after UVB treatment for RNA isolation and total protein extract preparation.
Wu X., Tommasi di Vignano A., Zhou Q., Michel-Dziunycz P.J., Bai F., Mi J., Qin J., Zu T, & Hofbauer G.F. (2018). The ARE-binding protein Tristetraprolin (TTP) is a novel target and mediator of calcineurin tumor suppressing function in the skin. PLoS Genetics, 14(5), e1007366.