Fig. 2

CNF1 induces oxidative stress and genetic instability in intestinal epithelial cells. (A) Representative CLSM micrographs of IEC-6 and HPCEC cells stained with anti-γH2AX (green) and anti-53BP1 (red) antibodies. Nuclei were counterstained with Hoechst (blue). Scale bars, 10 μm. (B) Bar plot showing fold change of γH2AX-positive nuclei and (C) bar plot showing fold change of 53BP1-positive nuclei in IEC-6 cells at different time points following exposure to CNF1. (D) Bar plot showing fold change of γH2AX-positive nuclei and (E) bar plot showing 53BP1-positive HPCEC nuclei at different time points following exposure to CNF1. One hundred nuclei at each experimental point were counted. (*p < 0.05; **p < 0.01; ***p < 0.001). (F) Representative fluorescence micrographs of IEC-6 cells stained with anti-pATR (green). Nuclei were counterstained with Hoechst (blue). Scale bars, 10 μm. (G) Western blot analysis of Chk1 and its phosphorylated form at the indicated time points. (H) Representative micrographs of Giemsa-stained IEC-6 nuclei showing: (a) normal anaphase; (b) bridge and (c) circular nucleus. (I) Bar plot showing the percentage of aberrant anaphases in untreated and in CNF1-treated IEC-6. One hundred nuclei for each experimental condition were analysed (***p < 0.001). (L) Representative micrographs and (M) bar plot showing the percentage of chromosome aberrations and polyploid cells in nuclei metaphases of IEC-6 cells treated with CNF1 as compared to untreated cells. (N) Bar plot showing the percentage of chromosome aberrations and polyploid cells after further 48 h of culture in the absence of CNF1 toxin. Two hundred metaphases were counted for each experimental condition (***p < 0.001). (O) Bar plots showing ROS concentration measured by EPR spectroscopy in IEC-6 and HPCEC cells exposed to 25 ρM CNF1 or to medium as control. (P) Representative fluorescence micrographs and bar plot showing the relative γH2AX amount in IEC-6 cells pre-treated with the anti-oxidant NAC (10 M) before CNF1 exposure. Nuclei are stained with Hoechst (blue). Scale bar: 10 μm. (*p < 0.05). (Q) Percentage of chromosome aberrations and polyploid cells of IEC-6 cells pre-treated with the anti-oxidant NAC before CNF1 exposure. Two hundred metaphases were counted for each experimental condition (***p < 0.001)