Fig. 9. Impaired tight junction sealing in response to hydrocortisone in EpH4 cells expressing constitutively activated PKN1. (A) Expression of constitutively activated PKN1 in EpH4 cells. RNA was prepared from cells stably transfected either with constitutively activated PKN1 (PKN) or vector alone (con) and subjected to RT-PCR using transgene specific primers (left panel). Bands for β-actin are shown as a control (right panel). (B) TER in PKN1 (PKN) and mock-transfected (con) cells either grown in the absence (–HC) or presence (+HC) of hydrocortisone. Data were derived from 12-15 filters in each group and experiments were performed on three independent occasions. Error bars depict the s.e.m. Hydrocortisone-stimulated PKN1 transfected cells were compared to stimulated mock-transfected cells using the Student's t-test (^***P<0.0005; ^**P<0.005). (C) Immunohistochemical detection of ZO-1 and occludin in PKN1 (PKN) and mock-transfected (con) cells. ZO-1 was visualized using a secondary antibody coupled to Cy3 whereas occludin was detected by a FITC-labeled secondary antibody. Nuclei were stained with DAPI.