Centromere reassembly during quiescence exit occurs in S phase but is independent of DNA replication. (A) Graph showing CENP-T 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 = 202, 336, 202, and 107 cells for cycling, quiescent, 24 h release EdU negative, and 24 h release EdU positive, respectively. * represents P < 0.05; P = 0.0348 between EdU negative and EdU positive using unpaired t test with Welch’s correction. Other pairwise comparisons can be found in Table S2. (B) Representative immunofluorescence images of cells released 24 h from quiescence. EdU-positive and -negative cells are shown. Cells were stained with CENP-T and anti-centromere (ACA) antibodies. Scale bar = 20 µm. (C) Graph showing CENP-T centromere intensity plotted against EdU intensity for each cell. CENP-T intensity is the average CENP-T 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 = 0.7092. n = 309 cells. (D) Graph showing CENP-O/P 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 = 214, 389, 185, and 114 cells for cycling, quiescent, 24 h release EdU negative, and 24 h release EdU positive, respectively. * represents P < 0.05; P = 0.0424 between EdU negative and EdU positive using unpaired t test with Welch’s correction. Other pairwise comparisons can be found in Table S2. (E) Representative immunofluorescence images of cells released 24 h from quiescence. EdU-positive and -negative cells are shown. Cells were stained with CENP-O/P and anti-centromere (ACA) antibodies. Scale bar = 20 µm. (F) Graph showing CENP-O/P centromere intensity plotted against EdU intensity for each cell. CENP-O/P intensity is the average CENP-O/P 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 = 0.753. n = 298 cells. (G) Representative immunofluorescence images of cells released from quiescence for 48 h with or without addition of 1 µM palbociclib. Cells were stained for either CENP-T or CENP-O/P and anti-centromere (ACA) antibodies. Scale bar = 10 µm. (H) Graph showing CENP-T 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 = 353, 429, 237, and 280 for cycling, quiescent, no palbociclib, and palbociclib, respectively. * represents P < 0.05; ns = not significant. P = 0.0184 between palbociclib and no palbociclib, P = 0.0177 between no palbociclib and quiescent, and P = 0.0878 between palbociclib and quiescent, using unpaired t test with Welch’s correction. Other pairwise comparisons can be found in Table S2. (I) Graph showing CENP-O/P 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 = 244, 355, 215, and 256 for cycling, quiescent, no palbociclib, and palbociclib, respectively. * represents P < 0.05, ns = not significant. P = 0.0286 between palbociclib and no palbociclib; P = 0.0278 between no palbociclib and quiescent, and P = 0.1902 between palbociclib and quiescent, using unpaired t test with Welch’s correction. Other pairwise comparisons can be found in Table S2. (J) Representative immunofluorescence images of cells released from quiescence for 48 h with or without addition of 2 mM thymidine. Cells were stained for either CENP-T or CENP-O/P and anti-centromere (ACA) antibodies. Scale bar = 10 µm. (K) Graph showing CENP-T 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 = 247, 343, 112, and 166 for cycling, quiescent, no thymidine, and thymidine, respectively. * represents P < 0.05; *** represents P < 0.001; ns = not significant. P = 0.1546 between thymidine and no thymidine, P = 0.0176 between no thymidine and quiescent, and P = 0.0002 between thymidine and quiescent, using unpaired t test with Welch’s correction. Other pairwise comparisons can be found in Table S2. (L) Graph showing CENP-O/P 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 = 260, 328, 149, and 190 for cycling, quiescent, no thymidine, and thymidine, respectively. * represents P < 0.05; ns = not significant. P = 0.1301 between thymidine and no thymidine, P = 0.0250 between no thymidine and quiescent, and P = 0.0368 between thymidine and quiescent, using unpaired t test with Welch’s correction. Other pairwise comparisons can be found in Table S2.