Basal p21 controls population heterogeneity in cycling and quiescent cell cycle states

Live-Cell Imaging Revealed p21-Dependent Population Heterogeneity.

To gauge cell cycle activity, we tracked the activity of CDK2 in single cells by using a fluorescent reporter consisting of the C-terminal CDK2 phosphorylation domain of DNA helicase B (DHB) fused to YFP (19). In G0 or G1 phase cells, the unphosphorylated reporter is located primarily in the nucleus (Fig. 1A and Fig. S1A). During the G1/S transition, S, and G2 phases, CDK2 phosphorylates the reporter, causing it to translocate from the nucleus to the cytoplasm (Movie S1). As a result, the cytoplasmic-to-nuclear ratio of DHB-YFP can be used to monitor CDK2 activity and cell cycle progression (12).

Using live-cell imaging to monitor the reporter in MCF10A cells, we could study growth factor-dependent (here synonymous with “mitogen-dependent”) heterogeneity in cell cycle activity. For typical experiments, researchers often desire maximal proliferation and therefore provide 20 ng/mL EGF and 5% (vol/vol) serum. However, this culture-optimized level of mitogenic stimulation is likely supraphysiological, and in comparison, human serum has been reported to contain 1–4 ng/mL EGF (20). In our experiments, when we supplemented cells with 20 ng/mL EGF and 5% (vol/vol) serum, almost all cells were actively cycling (Fig. 1B, Upper Right and Movie S1). In contrast, as expected, at a low level of stimulation (0.2 ng/mL EGF, 0.05% serum), all cells were quiescent (Fig. 1B, Upper Left) (when describing quiescence, here we do not distinguish G0 phase cells from those in prolonged G1). However, at an intermediate level of stimulation [5 ng/mL EGF, 1.25% (vol/vol) serum], we observed heterogeneity in cell cycle activity with 43% of cells in a cycling state and 57% of cells in a quiescent state (Fig. 1B, Upper Center and Movie S2).

In contrast, when we tracked the cell cycle activity of MCF10A cells deficient in p21, this heterogeneity was largely abrogated (Fig. 1B, Lower Center and Movie S3); 92% of cells were cycling. p21 is commonly known as a cell cycle inhibitor that is induced by the transcription factor p53 in response to DNA damage (Fig. 1C, WT+IR). However, even under normal, undamaged conditions, cells express a background or basal level of p21 (Fig. 1C, WT). We hypothesized that, in undamaged WT cells, the basal level of p21 was responsible for heterogeneity in cell cycle activity. This notion would be supported if expression of p21 independent of p53 restored population heterogeneity in p21-deficient cells. To test this hypothesis, we expressed p21 in cells with a p21-deficient genetic background using vectors where expression did not require p53 (i.e., constitutive expression). The ectopic p21 was expressed as a fusion with red fluorescent protein (RFP) to enable live single-cell fluorescence detection. The p21 was also fused to an estrogen receptor (ER) domain (RFP-p21-ER), and therefore, nuclear localization could be induced by the addition of 4-hydroxytamoxifen (4-OHT). Because the activity of p21 depends on its interactions with factors in the nucleus, we were thus able to induce the activity of p21 by addition of 4-OHT at the beginning of experiments. To achieve physiologically relevant levels of p21 expression, we used synthetic upstream open reading frames (uORFs) and different translation initiation site sequences (21, 22). We generated vectors that expressed the p21 fusion proteins over a broad range of levels (Fig. 1C and Fig. S1B). To approximate the relatively low basal level in undamaged WT cells, we chose a vector using two uORFs (Fig. 1C and Fig. S1 B–D, vector ACC/ACC/ACC). After ectopically expressing this low level of p21 in p21-deficient cells, we observed that, in the quiescent populations, p21 was sustained at high levels (Fig. 1D, Lower, red). In cycling populations, p21 was expressed at lower levels and exhibited oscillatory dynamics out of phase with CDK2 activity (Fig. 1D, Lower, blue and Fig. S1E). The single-cell dynamics, which indicated that p21 decreases at the start of S phase and increases at G2, were in line with previous studies that used synchronized populations to show the cell cycle-dependent regulation of p21 (23–25). Finally, at intermediate growth factor stimulation [5 ng/mL EGF, 1.25% (vol/vol) serum], ectopic p21 restored the heterogeneity in cell cycle activity exhibited by WT cells (Fig. 1D, Upper Center and Movie S4). Under these conditions, 52% of cells were actively cycling, whereas 48% remained quiescent.

Cellular Incorporation of BrdU Revealed a Range of Growth Factor Stimulation That Supported p21-Dependent Cell Cycle Heterogeneity.

Having established p21-dependent heterogeneity at an intermediate growth factor concentration using live-cell imaging, we next investigated the growth factor concentration range where quiescent and cycling states could coexist. We administered a 48-h pulse of BrdU to cells incubated in 12 different growth factor conditions and used flow cytometry to evaluate the fraction of each population that remained quiescent vs. actively cycling. Cells that did not incorporate any BrdU over a 48-h period were considered quiescent (Fig. S2A).

At low growth factor stimulation (between 0 ng/mL EGF, 0% serum and 3 ng/mL EGF, 0.75% serum), both WT and p21-deficient cells were largely quiescent (Fig. 2A, Top and Middle), and at high growth factor stimulation [between 8 ng/mL EGF, 2% (vol/vol) serum and 20 ng/mL EGF, 5% (vol/vol) serum], both WT and p21-deficient cells were mostly cycling (Fig. 2A, Top and Middle). However, over an intermediate range of stimulation [between 4 ng/mL EGF, 1% serum and 7 ng/mL EGF, 1.75% (vol/vol) serum], the WT population distributions were bimodal in BrdU incorporation, indicating that both quiescent and cycling states coexisted (Fig. 2A, Top). When the percentage of each population that remained quiescent was plotted against growth factor concentration, it could be seen that the response manifested as a graded change in the heterogeneity of the population (Fig. 2B). In contrast, p21-deficient populations exhibited significantly less heterogeneity at all growth factor levels. They responded to changes in growth factor stimulation with more switch-like behavior (Fig. 2 A, Middle and B) (represented by the steeper drop in the quiescent subpopulation)—when stimulation surpassed a threshold level (4 ng/mL EGF, 1% serum), the populations switched from being predominantly quiescent to predominantly cycling.

In line with results from the live-cell imaging experiments, in BrdU incorporation experiments, ectopic expression of p21 fused to GFP and the ER (GFP-p21-ER) also restored heterogeneity to p21-deficient populations (Fig. 2 A, Bottom and B). At the same intermediate growth factor stimulation that supported heterogeneity in WT cells [between 4 ng/mL EGF, 1% serum and 7 ng/mL EGF, 1.75% (vol/vol) serum], populations expressing ectopic p21 exhibited bimodality in BrdU incorporation. Ectopic p21 expression caused the sharp, switch-like response to growth factors observed in p21-deficient cells to become more graded, closely resembling the response of the WT population (Fig. 2B). This result further supported the notion that p21 bestows population heterogeneity in cycling and quiescent states.

Population Distributions in p21 Expression and Cell Cycle Phase Reflected Reversibility and Growth Factor-Dependent Heterogeneity in Cell States.

We next sought to determine whether the growth factor-dependent cycling and quiescent states were reversible. Reversibility would suggest that the heterogeneity in cell cycle activity was not caused by mutations or heritable epigenetic markings. We used flow cytometry to analyze population distributions in GFP-p21-ER and GFP-ER expression and cell cycle phase (Fig. S2 B–E). When flow cytometry analysis could not detect GFP-p21-ER expression in cells, we identified those populations as GFP-low. In the live-cell imaging and BrdU incorporation experiments, we expressed GFP-p21-ER using the ACC/ACC/ACC uORF-containing RNA leader sequence (Fig. 1C and Fig. S1B) to reproduce low basal expression levels. Although we could distinguish GFP-high and -low subpopulations, we could not confidently quantify their fractions in the total population because the GFP-p21-ER expression level was low. Thus, for analysis of GFP-p21-ER expression by flow cytometry, we used a leader that generated a moderate expression level (Fig. 1C, UCU/ACC and Fig. S1 B–D). We obtained similar results in experiments using the ACC/ACC/ACC and the UCU/ACC RNA leaders, but here, we have chosen to plot the results for the UCU/ACC leader.

To evaluate reversibility, we changed growth factor stimulation from maximal levels [20 ng/mL EGF, 5% (vol/vol) serum] to submaximal levels (0 ng/mL EGF, 0% serum and 4 ng/mL EGF, 1% serum) before returning to maximal stimulation. In line with our previous results, decreasing the growth factor stimulation levels shifted population distributions toward subpopulations in G1 with high p21 (Fig. S2B). When maximal stimulation was restored, the population distributions returned to ones dominated by cycling cells expressing low p21 (Fig. S2C), indicating reversibility in cycling and quiescent states.

We also sought to determine whether GFP-p21-ER expression levels were associated with distinct cell cycle-phase distributions. By staining DNA with propidium iodide (PI), we analyzed the cell cycle phase of each GFP-high and -low subpopulation. At all stimulation levels, the cells that highly expressed GFP-p21-ER were predominantly in G1 (2 N DNA content, and here, we do not distinguish G1 from G0) and, to a lesser extent, G2/M (4 N DNA content) (Fig. S2 B and C). No cells expressing high levels of GFP-p21-ER were found in S phase (0% 2 N < DNA < 4 N). In contrast, with intermediate and maximal growth factor stimulation, cells that were expressing low or undetectable levels of GFP-p21-ER were actively cycling, which was evidenced by the sizable population in S phase (∼30% 2 N < DNA < 4 N).

However, did the population distributions in GFP-p21-ER and cell cycle phase reflect population heterogeneity in cycling and quiescent states? Keep in mind that, even if all or nearly all cells in an unsynchronized population were cycling [e.g., the population under conditions of 20 ng/mL EGF and 5% (vol/vol) serum] (Fig. S2B), the cells in G1 and, to a lesser extent, G2/M would still express a high level of GFP-p21-ER. This high GFP-p21-ER expression occurs because, regardless of whether it is cycling, p21 is stabilized at these points during the cell cycle. However, a heterogeneous population consisting of a fraction of cells that is cycling and a fraction that is quiescent would exhibit an even greater percentage of cells with stabilized p21 (i.e., the GFP-high subpopulation). The existence of a quiescent G1-arrested population would further increase the percentage of GFP-high cells in the total population, because p21 would be stabilized in all of the quiescent cells as well as a fraction of the cycling cells. Thus, at an intermediate stimulation condition that supported heterogeneity in cell cycle activity (Fig. 1), the increased fraction of cells expressing high levels of GFP-p21-ER (4 ng/mL EGF, 1% serum; 41% GFP-high) over that of the maximal stimulation condition [20 ng/mL EGF, 5% (vol/vol) serum; 33% GFP-high] can be attributed to the quiescent subpopulation (Fig. S2B). We consider this increased fraction of GFP-high cells in the heterogeneous population to be telling and that, in general, flow cytometry analysis of p21 and cell cycle population distributions supported the results from live-cell tracking and BrdU incorporation experiments. As a control, we also expressed GFP-ER in p21^−/− cells. Under all conditions tested, GFP-ER expression was unimodal (Fig. S2 D and E). Thus, the growth factor-dependent regulation of GFP-p21-ER was likely dependent on the p21 domain.

Kinetic Model of Double-Negative Feedback Regulation Simulated Bistability in Cell Cycle States.

We postulated that the bistability in reversible cell states arose from double-negative feedback regulation involving CDK2 and p21 (Fig. 3 A and B). We created a kinetic model to show the potential bistability in levels of p21 and CDK2 activity at a single point in the cell cycle—namely, a period in G1 before the G1/S transition, when it is typically believed that cells have not committed to the next cell cycle (i.e., the restriction point).

The level of active CDK2 complexed with Cyclin E or Cyclin A (represented by CDK2_a in our equations) is affected by (i) growth factor stimulation, (ii) inhibition by p21, and (iii) positive feedback [a positive feedback loop was previously modeled by Yao et al. (17) and involved pRb, E2F, and Cyclin E]. The concentration of p21 is affected by its interaction with the Cyclin E-CDK2 complex through the action of SCF/Skp2 and other E3 ubiquitin ligase complexes (Fig. 3A and SI Text). The parameters used in the model are defined in Table S1.

We generated steady-state balance plots by solving for CDK2 activity over a range of p21 concentrations (Fig. 3B, purple lines) and then solving for p21 concentration over a range of CDK2 activity values (Fig. 3B, green lines) at three different growth factor concentrations. Each intersection of the CDK2 and p21 lines represented a steady state of the system. The system was monostable at both high and low growth factor concentrations (Fig. 3B, Left and Right). At intermediate growth factor concentration, three intersection points existed (Fig. 3B, Center), indicating that the system was bistable. The points corresponding to high p21/low CDK2 activity (Fig. 3B, Center, red circle) and low p21/high CDK2 activity (Fig. 3B, Center, blue circle) represented quiescent and cycling stable steady states, respectively.

Because our live-cell imaging experiments tracked p21, CDK2 activity, and cell cycle phase over multiple cell generations, we could examine levels of p21 and CDK2 activity within the G1 window described by our model. Under all stimulation conditions, we found that high p21/low CDK2 activity was associated with G1-arrested quiescent cells (Fig. 3C), whereas low p21/high CDK2 activity was associated with cycling cells in early G1, 3 h after mitosis and before the G1/S transition. Most importantly, under the intermediate stimulation condition [5 ng/mL EGF, 1.25% (vol/vol) serum], we found that coexisting quiescent and cycling states were also characterized by high p21/low CDK2 activity and low p21/high CDK2 activity, respectively. Therefore, together, modeling and experiments indicated that cell states were reflected by each cell’s position along a p21–CDK2 axis. Additionally, although there can be numerous other factors that distinguish G1 proliferating cells from G1 quiescent cells, the convergence between modeling and experimental results suggests that p21-dependent population heterogeneity can be explained, at least in part, by double-negative feedback regulation.

Although modeling p21 as the sole CKI was sufficient to describe the coexistence of two positions along a p21–CDK2 axis, there are two other kinase inhibitor proteins (KIPs), p27 and p57, regulating CDK2 that needed to be taken into account to appropriately model the CDK2 activity in our experiments (SI Text and Fig. S3). In particular, by including the contributions from p27 and p57, we were able to simulate the behavior of p21-deficient cells. Like p21, KIPs are regulated by CDK2 through the SCF/Skp2 E3 ubiquitin ligase, and they also inhibit CDK2 by binding to the CDK2-Cyclin E complex (13). In our model, the kinetic constants governing CDK2 and KIP regulation were the same as those involving p21.

Using this expanded model, we generated stimulus–response curves to evaluate bistability over a range of growth factor concentrations for three p21 generation rates. For each growth factor concentration, we plotted the corresponding steady-state CDK2 activity (Fig. S3 B and C) or CKI concentration (Fig. S3D). As with our previous experimental (Figs. 1B and 3C) and modeling results (Fig. 3B), when p21 was present in the system (k_gen,p21 = 1 or 2), the system was bistable at intermediate growth factor concentrations (Fig. S3 B, Center and Right, C, Center and Right, and D, Center and Right). In the p21-deficient case (k_gen,p21 = 0), the system remained monostable at all growth factor concentrations (Fig. S3 B, Left, C, Left, and D, Left).

Without the positive feedback on CDK2 (k₄ = 0), the change in CDK2 activity in p21-deficient cells was more graded (Fig. S3B, Left). However, when the model included the positive feedback on CDK2 (k₄ = 0.25), there was a sharp increase in CDK2 activity at a particular intermediate growth factor concentration in the p21-deficient case (Fig. S3C, Left). This increase also reflected the switch-like transition between quiescent and cycling states that we previously observed in p21-deficient cells at conditions between 3 ng/mL EGF and 0.75% serum and 4 ng/mL EGF and 1% serum (the sharp decrease in the percentage of quiescent cells in Fig. 2B). Thus, our model indicated that the switch-like behavior exhibited by p21-deficient cells in response to growth factor stimulation also depended on the positive feedback regulation of CDK2.

Cell Cycle Heterogeneity Is Dependent on WT p21 and SCF/Skp2 Activity.

Having proposed this double-negative feedback mechanism, we next sought to establish whether the negative regulation of p21 in our experiments depended on SCF/Skp2. When we inhibited p21 regulation using MLN-4924 (an inhibitor of Cullin-dependent E3 ligases) in p21-deficient cells expressing GFP-p21-ER, flow cytometry analysis of the p21 population distribution showed that a majority of the population had stabilized p21 and become quiescent, even at maximal stimulation (Fig. 4 A and B). We also evaluated expression of GFP-p21K6R-ER, a fusion protein containing a p21K6R mutant that could not be ubiquitinated by SCF/Skp2 at six known lysine targets (26). Across all levels of growth factor stimulation, populations predominantly showed high expression levels of p21K6R (Fig. 4C). Despite this stabilized expression of p21K6R, the cells still responded to varying growth factor stimulation. This result was consistent with previous reports that the mutant is not as active as WT p21 (26, 27). Because the mutant was stabilized at all levels of stimulation but diminished in its ability to inhibit cell cycle activity, the negative regulation of WT p21 (both endogenous p21 and GFP-p21-ER) was likely dependent on ubiquitination by SCF/Skp2 (or other Cullin-dependent complexes) and subsequent proteasomal degradation.

We then used our CDK2–p21 model to perturb the double-negative feedback regulation mechanism. Stabilizing p21 (setting k₂ to zero) (Fig. S4A, Left) caused the system to remain in a quiescent state (high p21/low CDK2 activity), even at high growth factor stimulation (Fig. S4A, Right). This result was consistent with our experimental observation that inhibiting SCF/Skp2 and other Cullin-dependent E3 ubiquitin ligase complexes with MLN-4924 caused populations to remain predominantly quiescent at high growth factor stimulation (Fig. 4 A and B). Because it was not possible to experimentally eliminate the regulation of CDK2 by p21 without also eliminating the regulation of p21 by SCF/Skp2 (26), we used our model (setting k₃ to zero) to simulate a p21 mutant unable to inhibit CDK2 but still negatively regulated by CDK2 through SCF/Skp2 (Fig. S4B, Left). This inhibition of p21 regulation caused the CDK2 activity on the steady-state balance plot to remain constant, resulting in monostability at all evaluated growth factor concentrations (Fig. S4B, Right). This result further supported the notion that the double-negative feedback mechanism plays a key role in generating bistability and population heterogeneity.

Expression Vectors.

The p21 gene was amplified from human cDNA by PCR. Monomeric GFP (EGFP A207K) was PCR-amplified from pEGFP-N1 (Clontech Laboratories), and ER domain was PCR-amplified from pBabe-Puro-OmoMyc-ER (a gift from Gerard Evan, University of California, San Francisco). Using these DNA fragments, we constructed the gene fusions encoding GFP-p21-ER and GFP-ER; using standard molecular biology techniques, the gene fusions were inserted into plasmids to generate pCru5-GFP-p21-ER and pCru5-GFP-ER, respectively. The fusions were then moved into pCru5-(UUU)_GGFP-IRES-Puro (22) using the EcoRI and NsiI restriction sites. The EcoRI-NotI fragment from this vector was then moved into the EcoRI-NotI sites of pCru5-GFP-IRES-mCherry vectors containing various RNA leader sequences and regulatory uORFs (22) to create pCru5-GFP-p21-ER-IRES-Puro and pCru5-GFP-ER-IRES-Puro. For live-cell imaging experiments, RFP (specifically, the fluorescent protein mCherry) was PCR-amplified from pCru5-GFP-IRES-mCherry and substituted for GFP to create pCru5-RFP-p21-ER-IRES-Puro. CSII-EF-DHB-YFP and CSII-EF-H2B-mTurquoise were constructed as previously described (12). GFP fusion constructs were used for flow cytometry experiments, because GFP detection is compatible with detection of PI staining. RFP fusion constructs were used for live-cell imaging experiments, because RFP detection is compatible with detection of the DHB-YFP CDK2 sensor.

Cell Culture.

MCF10A cells were maintained in DMEM/F12 media (11330–032; Life Technologies) supplemented with horse serum (H1138; Sigma-Aldrich), 2 mM glutamine, EGF (SRP3027; Sigma-Aldrich), 10 µg/mL insulin (I1882; Sigma-Aldrich), 500 ng/mL hydrocortisone (H0888; Sigma-Aldrich), 100 ng/mL cholera toxin (C8052; Sigma-Aldrich), 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 °C and 5% (vol/vol) CO₂. Full growth medium contained 5% (vol/vol) serum and 20 ng/mL EGF. During routine cell culture and throughout all experiments, cell populations were cultured at subconfluent concentrations to minimize contact inhibition and increased activation of p27.

To make a stable MCF10A WT line expressing the pCru5-IRES-Puro empty vector control and MCF10A p21^−/− lines expressing GFP-p21-ER, GFP-ER, RFP-p21-ER, GFP-p21K6R-ER, and the pCru5-IRES-Puro empty vector control, retroviral particles were made by cotransfecting the retroviral expression plasmids with pCL-Ampho (31) in 293T cells using calcium phosphate precipitation (CalPhos Mammalian Transfection Kit; Clontech Laboratories, Inc.). Supernatant containing viral particles was filtered and added to MCF10A cells along with 5 µg/mL polybrene (hexadimethrine bromide) for 18 h. The viral supernatant was tittered, such that each transduced cell received only one copy of the vector. Cells infected with pCru5-GFP-p21-ER, pCru5-GFP-ER, pCru5-RFP-p21-ER, and pCru5-IRES-Puro empty vector were selected with 1 µg/mL puromycin for at least 3 d and supplemented with 1 μg/mL puromycin during routine culture and 1 μg/mL puromycin and 500 nM 4-OHT throughout all experiments. Cultures infected with pCru5-GFP-p21K6R-ER were supplemented with 10 μg/mL blasticidin for at least 3 d to select for stable integration, and cells were maintained with 10 μg/mL blasticidin during routine cell culture and 10 μg/mL blasticidin and 500 nM 4-OHT throughout all experiments. To make MCF10A WT and p21^−/− lines that express DHB-YFP and H2B-mTurquoise, lentiviral particles were made by cotransfecting the lentiviral expression plasmids pCSII-EF-DHB-mVenus or pCSII-EF-H2B-mTurquoise (12) with pRSV-Rev, pHCMV-VSVG, and pMdlg in 293T cells using calcium phosphate precipitation. Supernatant containing viral particles was filtered and added to MCF10A cells along with 5 µg/mL polybrene (hexadimethrine bromide) for 18 h. Cells transduced with CSII-EF-H2B-mTurquoise and CSII-EF-DHB-mVenus lentivirus were sorted using a FACSAria (Beckton Dickinson) cell sorter to isolate a population expressing both constructs.

To determine the reversibility of quiescence and p21 stabilization at low growth factor stimulation, MCF10A p21^−/− cells expressing GFP-p21-ER or GFP-ER with a vector using the UCU/ACC uORF and RNA leader sequence (pCru5-UCU/ACC-GFP-p21-ER) were induced with 4-OHT for 24 h in media containing 20 ng/mL EGF and 5% (vol/vol) serum. Cells were then cultured with reduced growth factor stimulation (0 ng/mL EGF, 0% serum or 4 ng/mL EGF, 1% serum) for 72 h. Each culture was restored to full growth stimulation [20 ng/mL EGF, 5% (vol/vol) serum] for 72 h. For experiments examining the SCF/Skp2 dependence of p21 degradation, MCF10A p21^−/− cells expressing GFP-p21-ER were induced with 4-OHT for 24 h in media containing 20 ng/mL EGF and 5% (vol/vol) serum. Cells were then cultured with no growth factor stimulation (0 ng/mL EGF, 0% serum) for 72 h. Each culture was then restored to full growth stimulation [20 ng/mL EGF, 5% (vol/vol) serum] and concomitantly supplemented with either 0 or 1.38 µM MLN-4924 (A-1139; Active Biochem) cullin inhibitor for 48 h.

Flow Cytometry.

For DNA content analysis by PI staining, cells were trypsinized, rinsed with PBS, added to ice-cold 70% (vol/vol) ethanol, and stored at −20 °C overnight. Each sample was then rinsed with PBS and incubated in a buffer containing PI [PBS with 0.1% (vol/vol) Triton X-100, 0.2 mg/mL RNase A, and 20 μg/mL PI] for 30 min at room temperature.

To measure cell cycling and quiescent states by BrdU incorporation, cells were seeded in six-well plates (353046; Corning) and incubated in complete growth media containing 1 µg/mL puromycin and 500 nM 4-OHT for 24 h. Each well was then washed with PBS and incubated with media containing various concentrations of serum and EGF, 500 ng/mL hydrocortisone, 100 ng/mL cholera toxin, 1 µg/mL puromycin, and 500 nM 4-OHT for 72 h; 10 µM BrdU (B23151; Life Technologies) was added to each culture, and cells were incubated for an additional 48 h. Cells were then dissociated with 0.05% trypsin, and DMEM/F12 medium with 1% BSA was used to neutralize the trypsin. Cells were washed with PBS and incubated in 70% (vol/vol) ethanol overnight. After washing with PBS, DNA was denatured by treatment with PBS containing 0.1% Triton-X 100 (PBS-T) supplemented with 4 M HCl for 30 min. The acid was neutralized by washing two times with 0.1 M sodium tetraborate, and samples were incubated with PBS-T containing 1% BSA for 1 h for blocking; 1 µL mouse anti-BrdU antibody conjugated to AlexaFluor488 (clone MoBU1, B35139; Life Technologies) was added to each sample and incubated at 4 °C overnight before washing with PBS-T and incubating with PI staining solution for 30 min.

Cell populations were analyzed on an LSRII flow cytometer (Beckton Dickinson). GFP fluorescence intensity was determined using 488-nm excitation and a 525/50-band pass emission filter (the emission collection was centered at 525 nm with a total range of 50 nm) as well as 405-nm excitation and a 525/50-band pass filter. Flow cytometry data were analyzed using FlowJo (Tree Star).

Live-Cell Imaging.

MCF10A WT and p21^−/− cells expressing H2B-mTurquoise, DHB-YFP, and RFP-p21-ER (pCru5-ACC/ACC/ACC-RFP-p21-ER) were seeded in a 96-well plate (3603; Costar) in complete growth media containing 1 µg/mL puromycin and 500 nM 4-OHT and incubated for 24 h. Each well was washed two times with PBS, and 300 µL phenol red-free media containing various concentrations of serum and EGF, 500 ng/mL hydrocortisone, 100 ng/mL cholera toxin, 1 µg/mL puromycin, and 500 nM 4-OHT were added. Cells were incubated for 72 h before imaging for 48 h on an IXMicro microscope (Molecular Dynamics) with a heated stage and 5% (vol/vol) CO₂. A 10× objective was used, and total exposure time was kept below 900 ms. Images were analyzed using MATLAB (MathWorks).

Immunoblotting.

Immunoblotting was performed according to standard procedures. Lysates were loaded onto a 10% (vol/vol) Mini-Protean TGX Gel (Bio-Rad) and transferred to a PVDF membrane; p21 was detected using a primary anti-p21 mouse monoclonal antibody (2946; Cell Signaling Technology) and a secondary anti-mouse IgG conjugated to HRP (G21040; Molecular Probes). β-Actin was detected using a mouse monoclonal anti–β-actin antibody conjugated to HRP (A00730; Genscript). WesternBright ECL HRP substrate (K-12045-D50; Advansta) was used for HRP detection.

Modeling.

Computational analysis of the CDK2–p21 double-negative regulatory feedback system was performed using MATLAB (MathWorks).