Fig. 9. CNF-1 impairs the assembly of junctions in calcium switch assays. (A) T84 monolayers exposed to EGTA (2 mM) in calcium- and magnesium-free media were allowed to recover in complete culture media (containing calcium and serum) in the presence of CNF-1 (1 nM) or vehicle. TER was monitored over time and used to assess the recovery of cell-cell contact. Monolayers exhibited high resistances prior to EGTA treatment (#), which dropped to 10 .cm² following EGTA incubation (not shown). During the recovery period, control monolayers recovered high TER values of >700 .cm² after approximately 6 hours. By contrast, monolayers exposed to CNF-1 during recovery failed to reach TER values >170 .cm² (n=4) by 24 hours, indicating impaired junctional reassembly. (B) Occludin and E-cadherin staining was examined in monolayers immediately after EGTA treatment, and following recovery for 9 hours in complete media containing either CNF-1 (1 nM) or vehicle. Following EGTA treatment, both proteins were seen to move away from the lateral membrane into an intracellular compartment (a,b). During recovery in complete media with vehicle, both occludin (c) and E-cadherin (d) redistributed correctly into characteristic ring structures at the lateral membrane. By contrast, monolayers exposed to CNF-1 appeared to assemble only E-cadherin but not occludin into ring structures at the same time period (e,f).