5% BSA and 0.05% Tween20). Blots were washed repeatedly in washing LEE011 buffer (15 mM NaCl, 50 mM Tris-HCl, 0.05% Tween20; pH 7.6) and incubated for 1 h at room temperature with 0.1 μg/mL peroxidase-conjugated donkey anti-mouse IgG in blocking buffer. Peroxidase activity was detected using chemiluminescence substrate (Pierce) and recorded with a chemiluminescence detector

(Vilber Lourmat). Mouse anti-MEK1/2 (phosphorylated and non-phosphorylated), mouse anti-JNK (phosphorylated and non-phosphorylated) and mouse anti-p38 (phosphorylated and non-phosphorylated) were obtained from Cell Signaling Technology, Danvers, MA, USA For TransAm analysis, primary human keratinocytes were stimulated for 2 h with recombinant cytokines. Nuclear

extracts were generated with the Nuclear Extract Kit (Active Motif) and analyzed for activated transcription factors using TransAm Kits (Active Motif) according to the manufacturer’s protocols. For dual luciferase assays, primary human keratinocytes were grown to 70% confluence and transfected with two plasmids, MK-2206 molecular weight one containing the “Firefly Luciferase” under control of an AP-1-dependent promoter and a control plasmid expressing the “Renilla Luciferase” under the CMV promoter. The transfection was performed in presence of DMRIE-C (1, 2 -Dimyristyloxypropy l-3 – Dimethyl – Hydroxy – Ethyl–Ammoniumbromide plus Cholesterol) (Dual-Luciferase-Reporter Assay System, Promega). Eighteen hours after transfection, keratinocytes were stimulated for 48 h with recombinant cytokines. Concentration of CXCL-10, CXCL-11 and HBD-2 in cell-free supernatant of primary

human keratinocytes stimulated with 50 ng/mL IL-22, 50 ng/mL TNF-α or a combination of both were measured using commercially available sandwich ELISA kit according to the manufacturer’s instructions (CXCL-10, CXCL-11: R&D Systems, HBD-2: Phoenix Pharmaceuticals). C. albicans wild-type strain SC5314 was used for the infection of human oral keratinocytes (TR146, buccal carcinoma cell line) as described previously 33. C. albicans was grown on Sabouraud’s Oxymatrine dextrose agar (Difco) followed by two pre-cultures in 10 mL YPG (1% yeast extract, 2% peptone, 2% glucose) medium (Difco), first for 16 h at 25°C and then for 24 h at 37°C through orbital shaking. Human oral keratinocytes were cultured in DMEM medium supplemented with 10% FCS and 0.1% gentamicin solution (50 mg/mL) at 37°C and 5% CO2. For two-dimensional skin infection models, 30 000 human oral keratinocytes (TR146) were plated per well in 96-well plates in antibiotic and antimycotic free culture medium. Twenty-four hours after plating, cells were treated with 50 ng/mL TNF-α and IL-22 or Th22 supernatant. Each treatment was performed in triplicate. Keratinocytes were infected 30 min after treatment with a total amount of 3000 yeast cells (MOI 0.1).