Fig. 5.

ARHGEF11 plays a crucial role in the ZO-1–mediated remodeling of PJARs. (A–C) EpH4 cells depleted of ZO-1 and ZO-2 (ZO1KO·ZO2KD-EpH4) were cotransfected with a ZO-1 expression vector and control or ARHGEF11-specific siRNA, or transfected with ZO-1ΔCT (lacking aa 1520–1745). The expression of ZO-1 and ARHGEF11 was examined by Western blotting (A). (B) Distribution of the reexpressed WT or mutant ZO-1 was compared with that of myosin-IIB. (C) The number of cells exhibited the formation of PJAR represented by organized myosin-IIB as a thin line encircling cells was divided by the number of cells that were positive for reexpressed ZO-1. The percentage of the value was defined as rescued PJARs (y axis; data presented as mean ± SD; n = 3; *P < 0.001). (D) Recruitment of ARHGEF11 to TJs by WT or mutant ZO-1 was examined. ZO1KO·ZO2KD-EpH4 cells transfected with WT or ΔCT mutant ZO-1 were immunostained for ZO-1 and ARHGEF11. Arrows and arrowheads indicate cell–cell adhesion sites. (E) Effect of the simultaneous depletion of ARHGEF11 and ZO-2 on junction formation. ARHGEF11-specific siRNA was introduced into ZO2KD-EpH4 cells. The cells were stained for ARHGEF11 with myosin-IIB or occludin to investigate the organization of PJARs or TJs, respectively. Asterisks indicate ARHGEF11-depleted cells. The cells were analyzed 3 d after replating (A–E). (Scale bars: 10 μm.)