PMN traversal across S. suis strain 10 Δcps-infected PCPECs. PCPEC monolayers were stained for ZO-1 (red), actin (phalloidin, green), nuclei (DAPI, blue) and PMNs (SWC3a-FITC, yellow). Three-dimensional immunofluorescence images of S. suis strain 10 Δcps-infected PCPEC co-cultured with PMNs were reconstructed from 0.3 μm Apotome optical sections, using Zeiss software Inside 4D. Views from above (on the apical cell side) show different phases of PMN migration. The lower row represents side views (D, E, I, J, N, O). (A-E) PMN below TJ (right) and above TJ (left) and in the middle of a PCPEC (trancellular). The region where the left PMN migrated shows loose and diffuse actin staining at the upper PCPEC pole but the ZO-1 strands appears intact (white arrow). (F-J) PMN migration close to an interrupted ZO-1 strand (white arrow). (K-O) Transcellular PMN migration (left PMN, white arrow). The actin cytoskeleton is disarranged where the PMN exits the PCPEC (O). This figure is a representative example of three independent experiments that all gave similar results. Scale bar, as indicated.