Fig. 2. Regulation of ORC, Cdc6 and MCM proteins in cycling human cells and following reversible withdrawal into the quiescent state. (A) Immunoblot analysis of Orc2, Cdc6 and Mcm5 in chromatin-bound and soluble fractions of HeLa S3 cells during the indicated times after release from nocodazole arrest in metaphase (left panels) or double thymidine block at G1/S (right panels). Cell cycle synchronisation was verified by flow cytometry of isolated nuclei. Cells began to enter S phase 10 hours (*) after release from nocodazole arrest, and the majority of cells reached G2 phase by 8 hours () after release from a double thymidine block. The soluble fraction contains cytoplasmic and soluble nuclear proteins. (B) Immunoblot analysis of ORC, Cdc6 and MCM proteins in total cell extracts of asynchronously proliferating (AS) and contact-inhibited (G0) WI38 and NIH3T3 cells. Entry into quiescence was confirmed by BrdU labelling and antibody staining of asynchronously proliferating and contact-inhibited cultures (left panels; merged images of FITC (BrdU) and TOTO-3 (DNA) channels). (C) MCM protein expression in liver, an example of a stable tissue. The upper and lower panels show indirect immunoperoxidase staining of liver biopsies obtained from two patients undergoing liver transplantation with an anti-Mcm2 MAb (x160).