Fig. 2. Inhibition of -secretase enhances the effect of IL-1 on the contractile apparatus. Serum-starved cells were treated daily for 1 day (A) or 3 days (B,C) with IL-1 (10 ng/ml) or vehicle (control) and/or with DAPT (0.5 µM). (A) -Actin, SM-22, myocardin and calponin transcripts were assayed by RT-PCR. Results were expressed as a percentage of the mRNA level of control cells and represent the mean ± s.e.m. of three independent experiments. D+IL, DAPT plus IL-1 versus IL-1. *, P<0.05; **, P<0.01. (B) Immunostaining of -actin stress fibers was performed using a monoclonal antibody against -actin and a secondary antibody coupled to FITC (green-stain, 20x or 63x). Cell nuclei were stained with Hoechst (blue). Images are representative of four independent experiments. (C) 15 µg of total proteins were separated by electrophoresis (10% SDS PAGE). -Actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were immunodetected with appropriate antibodies. The autoradiogram is representative of three independent experiments. The histogram represents values obtained from scanning -actin bands normalized to that of GAPDH and are expressed as a percentage of control cells. C, control; IL, IL-1; D, DAPT; D+IL, DAPT plus IL-1 versus IL-1; **, P<0.01; ***, P<0.001; , P<0.05.