Figure 2.

Inhibition of cyclin A gene expression by APOE. (a) Cyclin A promoter activity in early-passage aortic SMCs was determined as described in Methods. Results show mean ± SEM, n = 3, *P < 0.001 as compared with cells stimulated with 10% FBS. C, control. (b) Quiescent SMCs were incubated for 48 hours with 10% FBS in the absence or presence of 2 μM APOA-I or APOE prior to analysis of cyclin A expression by immunofluorescence microscopy. DAPI staining shows cell nuclei. (c) Quiescent murine aortic SMCs treated with 10% FBS in the absence or presence of 2 μM APOE were collected, lysed, and analyzed by immunoblotting using Ab’s to cyclin D1, cyclin E, cyclin A, and cdk4 (loading control). (d) Aortic SMCs were transiently cotransfected with an eGFP expression vector and either a control (vector) or cyclin A expression vector. The effect of cyclin A expression on S-phase entry was determined in the absence or presence of 2 μM APOE or 2 μM APOA-I as described in Methods. Results show mean ± SEM, n = 2, *P < 0.001 as compared with cells stimulated with 10% FBS (vector). (e) A10 SMCs were treated as in c, collected at 20 hours, lysed, and analyzed by immunoblotting for cyclin A and actin (loading control). Cell lysates were incubated with anti-cyclin A, and the immunoprecipitates were used to assess in vitro kinase activity or analyzed by immunoblotting for associated cdk2.