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Fig. 1. Dissociation of epithelial junctions activates SRF-mediated transcription. (A) Dose-response curve of SRF activity. 1.5x106 MDCK cells were transiently transfected with SRF reporter p3DA-Luc (1.2 µg). Transfected cells were reseeded to form a confluent monolayer (600,000 per 1-cm diameter) and cultured in DMEM containing the physiological Ca2+ concentration (1.8 mM) for 24 hours. Then the medium was exchanged to the indicated amounts of Ca2+ and luciferase activity was measured 7 hours later. Shown is the relative luciferase activity normalised to protein amount. (B) Morphology and epithelial junction integrity of confluent monolayers after medium exchange to the Ca2+ amounts indicated in A. Cells were fixed and analysed by phase-contrast microscopy or immunostained for E-cadherin (DECMA-1). (C) SRF luciferase reporter activation on tissue culture plastic or in fully polarised cells grown on microporous membranes. Cells were either transfected with p3DA-Luc (3 x SRF) or a reporter containing SRF binding site mutations (mut.) and treated as in A. For comparison, cells were treated for 7 hours with 15% serum following 18 hours starvation (FCS) (left). To form a confluent monolayer 175,000 p3DA-Luc transfected cells were seeded on a Transwell filter (BD Falcon), and medium exchange was 24 hours later (right). (D) SRF activity in cells lacking E-cadherin expression. Rac V12 served as a positive control for the existence of a functional pathway in these cells. Transfection and stimulation was as before. E-cadherin expression (inset) in the three cell lines was determined by western blotting using anti-E-cadherin (clone 36, BD Transduction Laboratories). Mock, medium exchange with 1.8 mM Ca2+; –Ca2+, medium exchange with 0.02 mM Ca2+. Error bars indicate s.e.m. of three independent experiments. Stars indicate statistical significance at P
0.05 according to Student's unpaired t-test.
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