Fig. 5. Compound 3 prevents colibactin-induced genotoxicity in human cells.
a, Flow cytometry analysis of HeLa cells infected with NC101 and treated with 3. Cells were fixed in ethanol before staining with propidium iodide (PI) for DNA content. The increase in the population fraction with >2n DNA content indicates cell-cycle arrest. Top: raw histograms for PI fluorescence intensity in the population for one representative sample for each condition. Bottom: percentage of the population in G1 phase based on fitting histograms to the Watson cell-cycle analysis model50. Black symbols are individual replicates, bars show average value. All conditions were run in three biological replicates. Shading indicates the concentration of inhibitor. Gating strategy for flow cytometry is shown in Extended Data Fig. 7. b, Structure of two diastereomeric DNA adducts known to be derived from colibactin11. LC–MS detection of these adducts (M+H+ = m/z 540.1772) in hydrolyzed genomic DNA extracted from HeLa cells infected with NC101, three biological replicates are shown. Empty circles indicate sample was below the detection limit. For a and b ****P < 0.0001; *P < 0.05; NS, P > 0.05; one-way ANOVA and Dunnett’s multiple comparison test. c, Western blot for FANCD2 in HeLa cell extracts. All conditions were run in three biological replicates with one representative sample shown.