Stimulation of serum starved cells. BJ1 and RPE1 were serum starved for 24 h and then stimulated by addition of media with 10% serum. Samples were trypsinized, extracted with Triton X-100, fixed with MeOH, then stained and analyzed by flow cytometry. Data were preprocessed as in Additional file 3. Top panel:. the low Mcm6 G1 clusters (orange) decreased in frequency as a function of time, while the high Mcm6 (cyan) and PCNA positive S phase clusters (red-brown) increased. Lower panel: the fraction of cells residing in the low Mcm6 cluster (orange), high Mcm6 cluster, (cyan), and S phase (red-brown) were calculated as the percentage of G1 + S phase cells (Percent G1+S) and plotted as a function of time. The data were fit to constrained sigmoid equations with variable slopes (top = 100; bottom = 0). Kinetic analysis showed that the time to transit the high-Mcm6 state was 4.2 h for BJ1 and 2.1 h for RPE1 (arrows).