FIGURE 4.

Expression of a truncated, constitutively active ROCK1 overrides effects of anchorage deprivation; stabilizes CDC6, cyclin A, and D-type cyclins; and restores S6K1 Thr³⁸⁹ phosphorylation. A, logarithmically proliferating REF-Cdc6, Eker-Cdc6, REF-Cdc6-RK, and REF-Cdc6-aRK cells were cultured in methylcellulose medium, and cells were harvested every 12 h and analyzed for the indicated factors by immunoblotting. In parallel, the activities of Cdk2 and Cdk4 were assayed as described (29) (B), and the levels of cyclin D1, cyclin A, and E2F1 transcripts in REF-Cdc6 (open circles), Eker-Cdc6 (crosses), and REF-Cdc6-aRK (closed circles) cells were quantified by real time RT-PCR (C). Real time RT-PCR was performed on two or three independently isolated cell samples, and the data are shown as averages with S.D. bars. D, colony formation in soft agar. Logarithmically proliferating REF-Cdc6, Eker-Cdc6, REF-Cdc6-RK, and REF-Cdc6-aRK cells were cultured in DMEM containing 0.33% Noble agar layered on 0.5% bottom agar for 3 weeks with HeLa cells as a reference. Anc, anchorage; Cyc, cyclin.