Quantification of the colibactin-associated genotoxic effect by megalocytosis assay was performed as previously described [23]. Briefly, E. coli NC101 and K-12 strains from glycerol stocks were grown in LB at 37 °C, shaking overnight. Strains were sub-cultured in EMEM for 4 h. Caco-2 cells were dispensed (1 × 105 cells/well) in a 24 well tissue culture plate (Falcon) at 37 °C in a 5% CO2 atmosphere. After 24 h, Caco-2 cells were infected at a multiplicity of infection (MOI) of 50 with indicated E. coli strains. After 4 h of infection, the cells were washed at least three times with phosphate buffer saline (PBS) (Wisent Inc) and incubated for 72 h in cell culture medium supplemented with 200 μg/ml gentamicin (VWR). Cells were fixed with 4% paraformaldehyde (Thermo Fisher) for 15 min, washed and stained with 1 mM methylene blue (Sigma Aldrich). Pictures were taken under a Nikon Eclipse TE300 microscope (Nikon Healthcare, Québec, Canada) and images were acquired using the NIS-Elements BR4.00.03 software (200X magnification). Methylene blue extraction solution was used to quantify cell damage and megalocytosis at 660 nm absorbance (Spark® multimode microplate reader).

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