Ag1478

The cells of each LEC subline (5×10⁴) were seeded in 6-well plates in basal medium and incubated overnight. Then the cells were rinsed with PBS and fresh basal medium was added to the wells. Cell movement was recorded at 37°C and 5% CO₂ for 6 h at 5-min intervals using Leica DM IRE 2 microscope equipped with FW4000 software. The trajectories of 60 cells per experimental group were analyzed as previously described [30 (link)] in order to determine the basic cell motility parameters: the length of cell trajectory (LT), the average speed of cell movement (defined as LT divided by the time of recording), and the net displacement. In experiments testing the influence of growth factor receptor inhibitors on cell movement, the cells (2×10⁴) were seeded in 12-well plates in basal medium and incubated overnight. Then the cells were rinsed with PBS and incubated with fresh basal medium supplemented with: (i) EGFR inhibitor (AG-1478, Enzo Life Science; final concentration 1.5 μM), (ii) HER2 inhibitor (AG-825, Sigma; final concentration 1 nM), or (iii) both inhibitors. The control groups contained DMSO (vehicle), at the corresponding concentrations (0.01 or 0.001%). The recording of cell movement was started 1 h after the exposure of the cells to the inhibitors. Trajectories of 30 individual cells per experimental group were analyzed.

Dulbecco’s modified Eagle’s medium (DMEM)/F-12 and fetal bovine serum were purchased from Thermo Fisher Scientific (Grand Island, NY, USA). BioTrace^TM NT membrane was purchased from Pall Life Sciences (Ann Arbor, MI, USA). The enhanced chemiluminescence (ECL) Western blotting detection system was purchased from Perkin Elmer (Waltham, MA, USA). Anti-phospho-EGFR (Tyr¹¹⁷³, Cat#4407), anti-phospho-p38 MAPK (Thr¹⁸⁰/Tyr¹⁸², Cat#9211), anti-phospho-JNK1/2 (Thr¹⁸³/Tyr¹⁸⁵, Cat#9255), anti-phospho-FoxO1 (Ser²⁵⁶, Cat#9461), and anti-phospho-p65 (Ser⁵³⁶, Cat#3033) antibodies were purchased from Cell Signaling (Danvers, MA, USA). Antibodies of anti-TNFR1 (Cat#sc-52739), anti-TNFR2 (Cat#sc-8041), and p38 MAPK inhibitor (p38i) VIII were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-GAPDH (Cat#MCA-1D4) was purchased from EnCor Biotechnology (Gainesville, FL, USA). AG1478, SP600125, and Tanshinone IIA were purchased from Enzo Life Science (Farmingdale, NY, USA). AS1842856 was obtained from EMD Millipore (Billerica, MA, USA). Helenalin was purchased from Cayman Chemical (Ann Arbor, MI, USA). Recombinant human TNF-α protein was purchased from R&D Systems (Minneapolis, MN, USA). TRIzol reagent, enzymes, and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Foreskin-derived primary keratinocytes pooled from different individuals were purchased from PromoCell (Heidelberg, Germany) and cultured in Epilife medium (Life Technologies, Carlsbad, USA) at 37°C in a humidified atmosphere with 5% CO₂. For stimulation experiments with conidia and cytokines, keratinocytes were seeded in 12-well tissue culture plates (3.8 cm²/well, BD Biosciences) and used at 100% confluence. Cytokines used for stimulation were from PeproTech (Rocky Hill, NJ). The EGFR-blocking antibody cetuximab was obtained from Merck (Darmstadt, Germany) and AG1478 was purchased from Enzo Life Sciences (Farmingdale, NY). Typical strains of T. rubrum isolates were obtained from the Department of Dermatology, University Hospital of Schleswig-Holstein in Kiel, Germany isolated from tinea lesions of patients who have given written consent which was approved by the Institutional Review Board of the University of Kiel. Patient data were anonymized and de-identified prior to analysis. The strains were identified by established morphologic and physiologic criteria [9] . Microconidia were prepared as recently described [6] (link).