Fciii

Human lung fibroblasts (HFL‐1, CCL‐153, ATCC, Manassas, VA, USA) were expanded in Eagle's minimal essential medium (MEM, Biochrom, Berlin, Germany) supplemented with 1% penicillin–streptomycin (PEST), 1% glutamine, and 10% fetal clone serum (FCIII, Thermo Scientific, Waltham, MA) at 37°C, 5% CO₂. The cells were used in passages 14–20 with the majority of experiments performed in passages 18–20. Equivalent supplemented Dulbecco's modified Eagle's medium (DMEM, Sigma‐Aldrich, St Louis, MO) with 0.4% FCIII was used in general experimental conditions at 37°C, 10% CO₂, unless otherwise stated.
HFL‐1 cells were stimulated with 5‐HT (1 or 10 μmol/L, 5‐HT hydrochloride, Tocris, Bristol, UK) alone or in combination with TGF‐β1 (10 ng/ml) (R&D systems, Minneapolis, MN). TGF‐β1, known as a general inducer of remodeling (Westergren‐Thorsson et al. 2010), was added together with 5‐HT to stimulate fibroblast differentiation, mimicking physiologic remodeling. Fibroblasts were treated with the 5‐HT_2B receptor antagonists [dissolved in DMSO (Sigma‐Aldrich)] EXT5 (1 and 10 μmol/L) or EXT9 (1, 5, and 10 μmol/L) in combination with TGF‐β1 and 5‐HT (1 μmol/L), investigating potentially inhibitory effects of the antagonists.

Human SH-SY5Y neuroblastoma cells (ATCC, USA) were cultured in Dulbecco’s Modified Eagle’s/F12 medium (DMEM/F12, Sigma-Aldrich), 10% FCIII (VWR) and 1% antibiotic-antimycotic mixture (Sigma-Aldrich) in 5% CO₂ at 37 °C. In prior experiments, SH-SY5Y cells were incubated in a complete FCIII medium at a low serum content (1%) in presence of 15 μM RA (Sigma-Aldrich) for 72 h as previously described by us^{14 (link)} followed by treatment with PT, OT and AM (Sigma-Aldrich) for an additional 72 h using the concentrations described below. Cells incubated without thiamine antagonists were analyzed as control. Each experiment was repeated three times.