Figure 4. Effect of intracellular protein carbamylation on cell function and senescence. Confluent fibroblasts were incubated for 4 weeks at 37°C with DMEM + 0.5% (v/v) FBS without (control conditions) or with 20 mmol/L urea or 0.5 mmol/L cyanate. (a) Proliferation: cells were then seeded in 96-well plates at a density of 1,500 cells per well and incubated for 1, 2, 4 and 7 days with DMEM with 10% (v/v) FBS and carbamylating agents. Cell number was evaluated using a WST-1 assay by measuring absorbance at 450 nm. The data presented are means ± SEM (n=6) compared using the Mann-Whitney U test. No significant difference was found between the three conditions (control: dotted line,•; urea: grey line ■; cyanate: black line, ■). (b) Cell migration: cells were seeded in 24-well plates at a density of 15,000 cells per well and incubated for 24h at 37°C with DMEM containing 0.5% (v/v) FBS. Pictures of cells were taken every 30 min over the incubation period and each cell (n=58) was followed separately in order to calculate the migration speed. The data are presented as means ± SEM compared using the Mann-Whitney U test (*:p<0.05, **:p<0.01). (c) Expression of type I collagen mRNAs: at the end of the 4 weeks-incubation, RNA was isolated from confluent cells and then submitted to RT-qPCR analysis for evaluating the expression of COL1A1 and COL1A2 genes. Data represent the relative mRNA expression normalized to EEF1A1 gene and are expressed as means ± SEM (n=4). The Mann-Whitney U test was used to compare the three conditions: control (open bars), urea (grey bars) and cyanate (black bars). ns: not significant. (d) Senescence: cell senescence was determined by measuring the SA-β-galactosidase activity using a C12FDG fluorogenic substrate and by detection of senescent cells by flow cytometry. Each plot represents the results of 20,000 events acquired per condition. Incubation of cells with rotenone was used as a positive control of cell senescence whereas a negative control without addition of the fluorogenic substrate was performed.