Figure S3.
The centromere is rapidly reassembled upon cell cycle reentry. (A) Graph showing CENP-T centromere intensity level over time of quiescence exit. Each point indicates the average centromere intensity level for all centromeres in a single cell, adjusted for background. Intensity values were normalized to day 5. Red line represents the median, and blue points represent average of each replicate. Points were aggregated from three or more replicates. n = 613, 193, 182, 214, 456, and 336 cells for 0, 1, 2, 3, 5-day, and cycling time points, respectively. P values can be found in Table S2. (B) Graph showing CENP-O/P centromere intensity level over time of quiescence exit. Each point indicates the average centromere intensity level for all centromeres in a single cell, adjusted for background. Intensity values were normalized to day 5. Red line represents the median, and blue points represent average of each replicate. Points were aggregated from three or more replicates. n = 618, 200, 184, 218, 416, and 348 cells for 0, 1, 2, 3, 5-day, and cycling time points, respectively. P values can be found in Table S2. (C) Graph showing CENP-C centromere intensity level over time of quiescence exit. Each point indicates the average centromere intensity level for all centromeres in a single cell, adjusted for background. Intensity values were normalized to day 5. Red line represents the median, and blue points represent average of each replicate. Points were aggregated from three or more replicates. n = 698, 299, 335, 253, 440, and 346 cells for 0, 1, 2, 3, 5-day, and cycling time points, respectively. *** represents P = 0.0001. P values can be found in Table S2. (D) Graph showing CENP-K centromere intensity level over time of quiescence exit. Each point indicates the average centromere intensity level for all centromeres in a single cell, adjusted for background. Intensity values were normalized to day 5. Red line represents the median, and blue points represent average of each replicate. Points were aggregated from three or more replicates. n = 575, 148, 183, 175, 410, and 361 cells for 0, 1, 2, 3, 5-day, and cycling time points, respectively. P values can be found in Table S2. (E) Graph showing CENP-L centromere intensity level over time of quiescence exit. Each point indicates the average centromere intensity level for all centromeres in a single cell, adjusted for background. Intensity values were normalized to day 5. Red line represents the median, and blue points represent average of each replicate. Points were aggregated from three or more replicates. n = 554, 281, 213, 210, 462, and 275 cells for 0, 1, 2, 3, 5-day, and cycling time points, respectively. P values can be found in Table S2. (F) Graph showing CENP-A centromere intensity level over time of quiescence exit. Each point indicates the average centromere intensity level for all centromeres in a single cell, adjusted for background. Intensity values were normalized to day 5. Red line represents the median, and blue points represent average of each replicate. Points were aggregated from 5 replicates. n = 863, 423, 351, 416, 670, and 527 cells for 0, 1, 2, 3, 5-day, and cycling time points, respectively. P values can be found in Table S2. (G) Graph showing mRNA abundance for centromere components over time as cells exit quiescence. Fold change is calculated for each time point by comparing with cycling value. CT values were normalized to those of GAPDH before comparing released and cycling values. Graph shows at least three biological replicates, with three technical replicates each for each centromere mRNA. Bars represent mean ± SD. (H) Western blots of cells release from quiescence for the indicated amount of days. Blots were incubated with CENP-T and CENP-H antibodies. β-Actin is used as a loading control. (I) Graph showing CENP-C centromere intensity levels for different conditions: cycling, 7 days of quiescence, and either EdU-negative or EdU-positive cells from cells released 24 h from quiescence. Each point indicates the average centromere intensity level for all centromeres in a single cell, adjusted for background. Intensity values were normalized to cycling. Red line represents the median, and blue points represent average of each replicate. Points were aggregated from three replicates. n = 203, 304, 210, and 70 cells for cycling, quiescent, 24 h release EdU negative, and 24 h release EdU positive, respectively. * represents P < 0.05 and ns = not significant. P = 0.220 between EdU negative and EdU positive, P = 0.0259 between EdU positive and quiescent, and P = 0.0632 between EdU negative and quiescent using unpaired t test with Welch’s correction. Other pairwise comparisons can be found in Table S2. (J) Graph showing CENP-C centromere intensity plotted against EdU intensity for each cell. CENP-C intensity is the average CENP-C centromere intensity level for all centromeres in a cell, adjusted for background. EdU intensity is the mean EdU intensity in the nucleus of the same cell. Points were aggregated from three replicates. Values were normalized within each replicate. R2 = 0.1777. n = 272 cells. (K) Histogram showing the distribution of PI staining for cells release 48 h from quiescence without (orange) and with (pink) addition of palbociclib, as measured by flow cytometry (L) Graph showing the percentage of EdU-positive cells for the indicated conditions. Cells were released from quiescence into full serum media and incubated for 48 h in EdU with or without palbociclib or thymidine. Bars represent mean ± SD of four replicates for control, three replicates for thymidine, and two replicates for palbociclib. Mean of control is 92.03, mean of palbociclib is 0.3776, and mean of thymidine is 0. **** represents P < 0.0001, using unpaired t test with Welch’s correction. (M) Graph showing CENP-C centromere intensity for different conditions: cycling, quiescent, 48-h release from quiescence without palbociclib, and 48-h release from quiescence with palbociclib. Each point indicates the average centromere intensity level for all centromeres in a single cell, adjusted for background. Intensity values were normalized to cycling. Red line represents the median, and blue points represent average of each replicate. Points were aggregated from three replicates. n = 242, 346, 260, and 260 for cycling, quiescent, no palbociclib, and palbociclib, respectively. ** represents P < 0.01, ** represents P < 0.001, and ns = not significant. P = 0.0089 between palbociclib and no palbociclib, P = 0.0009 between no palbociclib and quiescent, and P = 0.0056 between palbociclib and quiescent, using unpaired t test with Welch’s correction. Other pairwise comparisons can be found in Table S2. (N) Graph showing CENP-C centromere intensity for different conditions: cycling, quiescent, 48-h release from quiescence without thymidine, and 48-h release from quiescence with thymidine. Each point indicates the average centromere intensity level for all centromeres in a single cell, adjusted for background. Intensity values were normalized to cycling. Red line represents the median, and blue points represent average of each replicate. Points were aggregated from three replicates. n = 186, 305, 141, and 101 for cycling, quiescent, no thymidine, and thymidine, respectively. * represents P < 0.05; ns = not significant. P = 0.4888 between thymidine and no thymidine, P = 0.0385 between no thymidine and quiescent, and P = 0.0488 between thymidine and quiescent, using unpaired t test with Welch’s correction. Other pairwise comparisons can be found in Table S2. (O) Western blot of 7-day quiescent cells, cells released from quiescence for 48 h in the presence of no drug, palbociclib, and thymidine, and asynchronous cycling cells. Blot was incubated with CENP-T, CENP-H, and CENP-C antibodies, and α-tubulin is used as a loading control. (P) Graph showing CENP-T centromere intensity in cycling cells treated with or without 2 mM thymidine for 48 h. Each point indicates the average of centromere intensity level adjusted for background for each replicate. Intensity values were normalized to control. Points were aggregated from three replicates. n = 894, 337 for control and thymidine, respectively. P = 0.0261 using unpaired t test with Welch’s correction. (Q) Graph showing CENP-O/P centromere intensity in cycling cells treated with or without 2 mM thymidine for 48 h. Each point indicates the average centromere intensity level for all centromeres in a single cell, adjusted for background. Intensity values were normalized to cycling. Red line represents the median. Points were aggregated from three replicates. n = 422, 197 for control and thymidine, respectively. P = 0.0206 using unpaired t test with Welch’s correction. Source data are available for this figure: SourceData FS3. Refer to the image caption for details. Panels A–F show centromere intensity for various proteins over time as cells exit quiescence. All plots use the same axes: Vertical Axis: Centromere Intensity (normalized units from negative 1 to 5). Horizontal Axis: Days out of quiescence (0, 1, 2, 3, 5, and cyc for cycling cells).Panel A: C E N P-T: This plot shows a sharp increase in C E N P-T intensity from day 0 to day 2, followed by a slight stabilization. There is a highly significant difference between day 0 and day 5, while the difference between day 5 and cycling cells is non-significant. Panel B: C E N P-O over P: Intensity for C E N P-O over P starts near zero at day 0 and peaks at day 2. The levels then slightly fluctuate through day 5 and cycling cells. A significant increase is noted between the initial and later stages, with no significant difference between day 5 and cycling cells. Panel C: C E N P-C: C E N P-C shows a more gradual, steady increase in intensity from day 0 through the cycling phase. While there is a significant overall increase compared to the baseline, the levels at day 5 are statistically similar (n s) to those in cycling cells. Panel D: C E N P-K: Similar to Panel A, C E N P-K levels rise sharply by day 2 and remain relatively stable through day 5 and the cycling phase. There is a high level of statistical significance between day 0 and day 5, but no significant difference between day 5 and cycling cells. Panel E: C E N P-L: This plot shows a sharp increase in C E N P-L centromere intensity by day 2 of quiescence exit, reaching a peak before stabilizing. There is a highly significant difference between day 0 and day 5, while the difference between day 5 and cycling cells is non-significant. Panel F: C E N P-A: C E N P-A displays relatively stable centromere intensity during the exit from quiescence compared to other proteins. While there is a moderately significant increase from day 0 to day 5, the levels at day 5 remain statistically similar to those in cycling cells. Panel G: q P C R: This line graph tracks the m R N A levels of C E N P T, C E N P N, C E N P A, and C E N P O. Most transcripts show a rapid increase that peaks between days 1 and 2 post-release before declining slightly toward cycling levels. Vertical axis shows normalized Abundance (released/cycling cells, 0.0 to 2.5). Horizontal axis shows days out of quiescence (0 to 5, and cyc). Panel H: Western Blot: This panel shows protein bands for C E N P-T and C E N P-H across different time points, with beta-actin as a loading control. The bands increase in thickness and intensity from the quiescent state through the various release days (1, 2, 3, 5). Vertical axis shows protein identity/molecular weight (k D a). The horizontal axis shows the experimental condition (quiescent and Release days 1, 2, 3, 5).Panel I: C E N P-C, 24-hour exit: This plot compares C E N P-C intensity across different cell states. It shows that, while quiescent cells have the lowest intensity, E d U-positive cells (undergoing D N A replication) show significantly higher C E N P-C loading than E d U-negative cells at the 24-hour mark. Vertical axis shows Centromere intensity (0.0 to 2.0). Horizontal axis show cell population (cycling, quiescent, E d U negative, EdU positive).Panel J: Correlation: This scatter plot shows the relationship between D N A replication and protein loading. A regression line indicates a weak positive correlation (R 2 equals 0.1777), suggesting that as E d U intensity increases, CENP-C intensity tends to rise. The vertical axis shows C E N P-C intensity. Horizontal axis shows E d U intensity. Panel K: Flow Cytometry: These histograms show DNA content distribution (P I-A). The pink plot (plus palbociclib) shows a single sharp peak indicating G 1 arrest, while the orange plot (48 h release) shows a broader distribution including S and G 2/M phases. Vertical axis shows Cell count/frequency.Horizontal Axis: P I-A (D N A content). Panel L: 48 hour release: This bar graph quantifies the percentage of cells successfully replicating D N A. The control group shows nearly 100 percent E d U positivity, while Palbociclib and thymidine treatments show a near-total reduction in D N A synthesis. Vertical axis shows percentage E d U positive (0 to 100). Horizontal axis shows treatment group (control, Palbociclib, thymidine). Panel M: C E N P-C, 48 hour exit (Palbociclib): This plot compares C E N P-C intensity after 48 hours of release from quiescence with and without Palbociclib treatment. While intensity recovered to cycling levels in the no Palb group, Palbociclib-treated cells show a significant reduction in C E N P-C loading, though levels remain slightly higher than the baseline quiescent state. Vertical axis shows Centromere Intensity (normalized units from negative 1 to 4). Horizontal axis shows cell population (cycling, quiescent, no Palb, Palb). Panel N: C E N P-C, 48-hour exit (Thymidine): Similar to Panel M, this plot examines the effect of thymidine on C E N P-C recovery. Unlike Palbociclib, thymidine-treated cells show C E N P-C intensity levels that are statistically similar to the no thymidine release group, with both being significantly higher than the quiescent state. The vertical axis shows Centromere Intensity (normalized units from negative 1 to 4). The horizontal axis shows cell population (cycling, quiescent, no thymidine, thymidine). Panel O: Western Blot (Inhibition Analysis): This panel provides a qualitative assessment of total protein levels for C E N P-T, C E N P-H, and C E N P-C across various inhibition conditions, using alpha-tubulin as a loading control. The results show that protein abundance generally increases upon release (no drug) but varies depending on the specific inhibitor used. Vertical axis shows Protein identity/molecular weight (k D a). The horizontal axis shows Experimental condition (quiescent, no drug, plus palbociclib, plus thymidine, cycling). Panel P: C E N P-T (Bar Graph): This graph quantifies the centromere intensity of C E N P-T, specifically comparing control cells to those treated with thymidine. There is a significant increase in C E N P-T loading in the thymidine-treated group compared to the control. Vertical Axis shows Centromere Intensity (normalized units from 0.0 to 2.5). Horizontal Axis show Treatment group (control, thymidine). Panel Q: C E N P-O/P (Bar Graph): This graph quantifies C E N P-O/P centromere intensity under thymidine treatment. Similar to C E N P-T, there is a significant increase in the intensity of C E N P-O/P at the centromere in thymidine-treated cells compared to the control group. The vertical axis shows Centromere Intensity (normalized units from 0 to 4). The horizontal axis shows the treatment group (control, thymidine). All data is approximate.

The centromere is rapidly reassembled upon cell cycle reentry. (A) Graph showing CENP-T centromere intensity level over time of quiescence exit. Each point indicates the average centromere intensity level for all centromeres in a single cell, adjusted for background. Intensity values were normalized to day 5. Red line represents the median, and blue points represent average of each replicate. Points were aggregated from three or more replicates. n = 613, 193, 182, 214, 456, and 336 cells for 0, 1, 2, 3, 5-day, and cycling time points, respectively. P values can be found in Table S2. (B) Graph showing CENP-O/P centromere intensity level over time of quiescence exit. Each point indicates the average centromere intensity level for all centromeres in a single cell, adjusted for background. Intensity values were normalized to day 5. Red line represents the median, and blue points represent average of each replicate. Points were aggregated from three or more replicates. n = 618, 200, 184, 218, 416, and 348 cells for 0, 1, 2, 3, 5-day, and cycling time points, respectively. P values can be found in Table S2. (C) Graph showing CENP-C centromere intensity level over time of quiescence exit. Each point indicates the average centromere intensity level for all centromeres in a single cell, adjusted for background. Intensity values were normalized to day 5. Red line represents the median, and blue points represent average of each replicate. Points were aggregated from three or more replicates. n = 698, 299, 335, 253, 440, and 346 cells for 0, 1, 2, 3, 5-day, and cycling time points, respectively. *** represents P = 0.0001. P values can be found in Table S2. (D) Graph showing CENP-K centromere intensity level over time of quiescence exit. Each point indicates the average centromere intensity level for all centromeres in a single cell, adjusted for background. Intensity values were normalized to day 5. Red line represents the median, and blue points represent average of each replicate. Points were aggregated from three or more replicates. n = 575, 148, 183, 175, 410, and 361 cells for 0, 1, 2, 3, 5-day, and cycling time points, respectively. P values can be found in Table S2. (E) Graph showing CENP-L centromere intensity level over time of quiescence exit. Each point indicates the average centromere intensity level for all centromeres in a single cell, adjusted for background. Intensity values were normalized to day 5. Red line represents the median, and blue points represent average of each replicate. Points were aggregated from three or more replicates. n = 554, 281, 213, 210, 462, and 275 cells for 0, 1, 2, 3, 5-day, and cycling time points, respectively. P values can be found in Table S2. (F) Graph showing CENP-A centromere intensity level over time of quiescence exit. Each point indicates the average centromere intensity level for all centromeres in a single cell, adjusted for background. Intensity values were normalized to day 5. Red line represents the median, and blue points represent average of each replicate. Points were aggregated from 5 replicates. n = 863, 423, 351, 416, 670, and 527 cells for 0, 1, 2, 3, 5-day, and cycling time points, respectively. P values can be found in Table S2. (G) Graph showing mRNA abundance for centromere components over time as cells exit quiescence. Fold change is calculated for each time point by comparing with cycling value. CT values were normalized to those of GAPDH before comparing released and cycling values. Graph shows at least three biological replicates, with three technical replicates each for each centromere mRNA. Bars represent mean ± SD. (H) Western blots of cells release from quiescence for the indicated amount of days. Blots were incubated with CENP-T and CENP-H antibodies. β-Actin is used as a loading control. (I) Graph showing CENP-C centromere intensity levels for different conditions: cycling, 7 days of quiescence, and either EdU-negative or EdU-positive cells from cells released 24 h from quiescence. Each point indicates the average centromere intensity level for all centromeres in a single cell, adjusted for background. Intensity values were normalized to cycling. Red line represents the median, and blue points represent average of each replicate. Points were aggregated from three replicates. n = 203, 304, 210, and 70 cells for cycling, quiescent, 24 h release EdU negative, and 24 h release EdU positive, respectively. * represents P < 0.05 and ns = not significant. P = 0.220 between EdU negative and EdU positive, P = 0.0259 between EdU positive and quiescent, and P = 0.0632 between EdU negative and quiescent using unpaired t test with Welch’s correction. Other pairwise comparisons can be found in Table S2. (J) Graph showing CENP-C centromere intensity plotted against EdU intensity for each cell. CENP-C intensity is the average CENP-C centromere intensity level for all centromeres in a cell, adjusted for background. EdU intensity is the mean EdU intensity in the nucleus of the same cell. Points were aggregated from three replicates. Values were normalized within each replicate. R2 = 0.1777. n = 272 cells. (K) Histogram showing the distribution of PI staining for cells release 48 h from quiescence without (orange) and with (pink) addition of palbociclib, as measured by flow cytometry (L) Graph showing the percentage of EdU-positive cells for the indicated conditions. Cells were released from quiescence into full serum media and incubated for 48 h in EdU with or without palbociclib or thymidine. Bars represent mean ± SD of four replicates for control, three replicates for thymidine, and two replicates for palbociclib. Mean of control is 92.03, mean of palbociclib is 0.3776, and mean of thymidine is 0. **** represents P < 0.0001, using unpaired t test with Welch’s correction. (M) Graph showing CENP-C centromere intensity for different conditions: cycling, quiescent, 48-h release from quiescence without palbociclib, and 48-h release from quiescence with palbociclib. Each point indicates the average centromere intensity level for all centromeres in a single cell, adjusted for background. Intensity values were normalized to cycling. Red line represents the median, and blue points represent average of each replicate. Points were aggregated from three replicates. n = 242, 346, 260, and 260 for cycling, quiescent, no palbociclib, and palbociclib, respectively. ** represents P < 0.01, ** represents P < 0.001, and ns = not significant. P = 0.0089 between palbociclib and no palbociclib, P = 0.0009 between no palbociclib and quiescent, and P = 0.0056 between palbociclib and quiescent, using unpaired t test with Welch’s correction. Other pairwise comparisons can be found in Table S2. (N) Graph showing CENP-C centromere intensity for different conditions: cycling, quiescent, 48-h release from quiescence without thymidine, and 48-h release from quiescence with thymidine. Each point indicates the average centromere intensity level for all centromeres in a single cell, adjusted for background. Intensity values were normalized to cycling. Red line represents the median, and blue points represent average of each replicate. Points were aggregated from three replicates. n = 186, 305, 141, and 101 for cycling, quiescent, no thymidine, and thymidine, respectively. * represents P < 0.05; ns = not significant. P = 0.4888 between thymidine and no thymidine, P = 0.0385 between no thymidine and quiescent, and P = 0.0488 between thymidine and quiescent, using unpaired t test with Welch’s correction. Other pairwise comparisons can be found in Table S2. (O) Western blot of 7-day quiescent cells, cells released from quiescence for 48 h in the presence of no drug, palbociclib, and thymidine, and asynchronous cycling cells. Blot was incubated with CENP-T, CENP-H, and CENP-C antibodies, and α-tubulin is used as a loading control. (P) Graph showing CENP-T centromere intensity in cycling cells treated with or without 2 mM thymidine for 48 h. Each point indicates the average of centromere intensity level adjusted for background for each replicate. Intensity values were normalized to control. Points were aggregated from three replicates. n = 894, 337 for control and thymidine, respectively. P = 0.0261 using unpaired t test with Welch’s correction. (Q) Graph showing CENP-O/P centromere intensity in cycling cells treated with or without 2 mM thymidine for 48 h. Each point indicates the average centromere intensity level for all centromeres in a single cell, adjusted for background. Intensity values were normalized to cycling. Red line represents the median. Points were aggregated from three replicates. n = 422, 197 for control and thymidine, respectively. P = 0.0206 using unpaired t test with Welch’s correction. Source data are available for this figure: SourceData FS3.

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