Figure 8. Recombinant and PMN-MP associated MPO inhibits IEC proliferation by inducing cell cycle arrest.

The effect of soluble and MP-associated MPO on IEC proliferation was assayed using cell cycle analysis and Edu incorporation assay. (A) Propidium iodide staining and flow cytometry was used to perform cell cycle analyses of subconfluent IECs under the specified conditions. Treatment with either recombinant MPO or PMN-MPs with the associated MPO in the presence of H₂O₂ resulted in a significant accumulation of IECs in G2/M phase, suggesting cell cycle arrest. * p<0.05, ** (p<0.01), *** (p<0.001). Data are shown as averages of 3 independent experiments. (B, C) Proliferation of the leading edge cells in scratch wounded IEC monolayers was assessed by Edu incorporation. (B) At least 5 random fields per each condition were analyzed; the data are presented as % proliferating (nuclei stained in purple, EdU) with respect to total cells (nuclei stained in blue, topro-3) in the field. ^ns not significant. * different from control (p<0.05), ** (p<0.01), *** (p<0.001). (C) Images representative of three independent experiments show significantly decreased number of proliferating cells at the sites of closing wounds (wound edge) following treatment with either recombinant or PMN-MP associated MPO in presence of H₂O₂. Red (Edu, proliferative cells), Blue (Dapi, nuclei stain).