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Movie S1. CDK2 Activity in MCF10A Cells Emerging from Mitogen Starvation, Related to Figure 2.
MCF10A cells expressing DHB-Ven were starved in GM-GFS for 40 hr and then stimulated with full growth media at the start of the movie. Cells were imaged every 12 min for 30 hr with a 20× objective. The green arrow marks the cell whose CDK2 activity is plotted underneath. The time of S phase entry was scored using Cer-Cdt1 (data not shown) and the time of anaphase was scored used H2B-Chy (data not shown).
Movie S2. CDK2 Activity in cycling MCF10A Cells, Related to Figure 3.
MCF10A cells expressing DHB-Ven were imaged in full growth media every 12 min for 24 hr with a 20× objective. The blue arrow marks a cell that enters the CDK2inc state after mitosis, whereas the red arrow marks a cell that enters the CDK2low state after mitosis. This cell emerges from this state at the end of the movie by building up CDK2 activity. The two cells’ CDK2 activity through time is plotted underneath. The time of anaphase was scored used H2B-Chy (data not shown).
Movie S3. Time-Lapse Imaging of CDK2 Activity Followed by Immunofluorescence, Related to Figure 4.
MCF10A cells expressing H2B-Turq (red pseudo-color) and DHB-Ven (green pseudo-color) were imaged every 12 min for 10 hr with a 20× objective (frames 1–50). Each nucleus was assigned a unique number for the duration of the movie. At the end of the time-lapse imaging period, cells were immediately fixed in formaldehyde and stained with a primary antibody to phospho-Rb at Serine 807/811. This antibody signal was visualized using a secondary antibody conjugated to Alexa Fluor 647 and imaged with a far red filter (blue pseudo-color, frame 51). A custom jitter correction script was used to align all nuclei before and after antibody staining (see Extended Experimental Procedures), which enabled us to map each cell in the immunofluorescence image back to same cell recorded in the CDK2 time-lapse analysis.
Movie S4. In Response to MEK Inhibition, CDK2inc Cells Complete the Current Cell Cycle and Then Enter the CDK2low State after the Subsequent Mitosis, Related to Figure 6.
MCF10A cells expressing DHB-Ven (green pseudo-color) were imaged every 12 min for 48 hr with a 10× objective. After 8 hr of imaging, cells were treated with 100 nM MEK inhibitor (PD032591). Cells were in the presence of the MEK inhibitor for the remaining 40 hr of imaging. After drug addition, cells proceed through the cell cycle at the typical rate, undergo mitosis, and then enter the CDK2low state, characterized by nuclear DHB-Ven. Scale bar (frame 1), 20 microns.
Spencer, S. L. et al. The proliferation-quiescence decision is controlled by a bifurcation in CDK2 activity at mitotic exit. Cell 155, 369–383 (2013).