Cell culture and proliferation assay

The human lung carcinoma epithelial cell line A549 (American type culture collection, CCL-185) was maintained by serial passage in DMEM/F-12 (Sigma-Aldrich Corp., St. Louis, MI, USA) with 10% FBS (Sigma-Aldrich Corp., St. Louis, MI, USA). For the proliferation assay, cells were seeded at a concentration of 1×10⁵ cells/mL in Corning 96-well black polystyrene microplates with clear bottom (Fischer Scientific, Illkirch, France) and cultured at 37°C and 5% CO₂. Cells were challenged with 5 μL supernatant (1:20 dilution) collected from 5FC exposed wt, ΔfcyA, Δuprt and ΔuprtΔurkA liquid cultures. Supernatant was added either immediately after seeding or after cells adhered, which consistently occurred after 6 h. Proliferation of A549 cells was monitored over the duration of 48 h with the IncuCyte S3 Live-Cell Analysis System. Four images per well were acquired every 2 h. For A549 cells, the magnification required with the S3/SX1 G/R Optical Module was 10×. Cell confluence was determined using the Basic Analyzer software of the IncuCyte S3 for phase imaging.

Resazurin-based assay was performed as previously described [36] with few modifications. For this, cells were incubated in the presence of supernatants for 48 h before adding 50 μM resazurin for another 4 h. Subsequently, fluorescence measurements were performed using the CLARIOstar Plus microplate reader (BMG LABTECH, Ortenberg, Germany) with excitation at 544 nm and emission at 590 nm. All experiments have been performed in triplicates.