Assessment of cell cycle progression under synchronizing conditions. a, b) Flow cytometry analysis of DNA-stained cell cultures grown under N+, ND, and NR conditions. Samples of cells grown under synchronizing conditions were fixed and stained with SYTOX Green prior to flow cytometry analysis. Summary of DNA content (a) of cells at non-S/M (ZT0-10, ZT24) and S/M phases (ZT12-16) of N+ condition as well as cells under ND (ND0, 6, 12, and 36) and NR (NR6 and 12) conditions. Y-axis: average population (%) of cells carrying single (green), ≥2 (orange), or undetermined (gray) copies of chromosomes. n = 2 for each sample. Example plot (b) showing particle size (FSC-A; Forward scatter) vs DNA content (FITC-A) for samples at ZT6. Each dot represents noncell particle (black) or a cell carrying a single (green), 2 (orange), >2 (blue), or undetermined (brown and cyan) copies of the genome. One of the 2 replicates (n = 2) for each sample is presented. See also Supplementary Fig. 1. c, d) Cell morphology of wild type, CHT7-HA::cht7, and cht7 cells at ZT6. Two biological replicates (r1 and r2) for each strain were observed under the microscope and summarized in (c). n: numbers of cells observed. Y-axis: population (%) of cells of type 1 (green), 2 (orange), 3 (gray), 4 (yellow), and 5 (blue). The representative graphs and definition of each cell type are shown in (d).