Fig. 10. Stimulation of tight junction sealing in EpH4 cells expressing a dominant-negative PKN1 mutant. (A) Expression of the dominant-negative PKN1 mutant PKN1 T774A (PKNT774A) in EpH4 cells. RNA prepared from cells stably transfected either with a construct encoding the dominant-negative PKN1 mutant PKN1 T774A or vector alone was subjected to RT-PCR using primers for the house keeping gene Gapdh (left panel) or the dominant-negative PKN1 mutant (right panel). In lanes marked with –RT, reverse transcriptase was omitted during RT-PCR as a control for DNA contamination; + indicates plasmid DNA used as positive control. (B) TER in EpH4 cells transfected with PKN1 T774A (PKN1 d.n.) or vector alone and either grown in the absence (–HC) or presence (+HC) of hydrocortisone. Data were derived from eight filters in each group and experiments were performed on three independent occasions. Error bars depict the s.e.m. and cells expressing PKN1 T774A were compared with control cells growing under the same conditions using the Student's t-test. Significant differences with P<0.05 are marked with # for cells grown in the absence of hydrocortisone and ^* for cells stimulated with hydrocortisone.