IECs were cocultured with either freshly isolated PMNs or PMN-MPs (ratio of 2 PMNs, or MPs derived from 4 PMNs, to 1 IEC). (A) FACS analysis of IEC cell cycle with or without PMN-MP treatment and the addition of ROS scavenger NAC (2 mM, 24 hours, n = 4, **P < 0.01). (B) Detection of DSBs in PMN- or PMN-MP–treated IECs with or without NAC (2 mM, 24 hours) or MnTBAP (100 μM, 24 hours) by COMET assay. Representative images of normal and DSB-positive nuclei (right panel, n = 3, **P < 0.01). (C) ELISA detecting 8-oxoG in genomic DNA isolated from IECs treated with PMNs, PMN-MPs with or without NAC (2 mM), or MnTBAP (100 μM) (24 hours, n = 4, ***P < 0.001). (D) Proliferation rate of PMN-MP–treated HCT116 and CaCo2 was monitored over indicated times (n = 4, **P < 0.01). (E and F) PMN-MP effect on IEC replication was assessed by single-labeled DNA fiber analysis. Following 24 hours of treatment, BrdU was added to IECs for 40 minutes or 2 hours, and its incorporation was quantified by immunofluorescence labeling from images as shown in E. The length of more than 400 fibers per condition was analyzed for each cell type (n = 4, **P < 0.01). (G–I) For analysis of IEC replication fork stability, DNA fibers were extracted (as described in Supplemental Methods) and double labeled according to schematics depicted in G, where CldU incorporation (H, green) indicates replication before fork stalling and IdU (H, red) indicates recovered replication forks. (H) Representative images of double-labeled fibers of control and PMN-MP–treated cells. (I) The lengths of separated tracks for each cell type were analyzed in over 400 fibers/condition (n = 4, **P < 0.01). Two-tailed Student’s t test was used for statistical analyses (P values). Data are mean ± SD from at least 3 independent experiments.