Figure 1.

cdc6 mRNA levels are downregulated in differentiated HEL(Esg+) cells. (A) HEL and HEL(Esg+) were cultured for 48 h in the absence (−) or in the presence (+) of 10⁻⁸ M TPA for 48 h. Total RNA was extracted and analyzed by northern blotting with full-length cdc6 and 28S rRNA-specific cDNA probes. Quantification of cdc6 mRNA signals normalized by 28S RNA is also shown (dark bars, 3.3 kb specie and light bars, 2.5 kb specie). (B) Cells were cultured as in (A), and then treated for the indicated times with 5 μg/ml Act D. RNA extraction and analysis was performed as in (A). (C) HEL and HEL(Esg+) cells were seeded in triplicate wells and cultured in the absence (closed circles) or in the presence (open triangles) of TPA. At the indicated times, cells were harvested and viable cells were counted as presented in Materials and Methods. (D) Cells were cultured as in (A) and nuclear extracts were isolated, subjected to SDS–PAGE and analyzed by western blotting with anti-Cdc6 antibody. A portion of total protein staining of transferred gel is shown as a loading control.