Fig. 3: Nucleotide imbalance impairs S phase progression.

a, Approach using flow cytometry to assess cell cycle phase by DNA content (as determined by propidium iodide staining) and EdU incorporation. b, Cell cycle distribution of A549 cells cultured with the indicated concentration of guanine (G) for 24 h. c, Cell cycle distribution of A549 cells treated with or without 200 µM G with or without 200 µM adenine (A) for 24 or 48 h. d, Approach to assess S phase progression. After pulsing cells with EdU, cell cycle progression of EdU-positive and EdU-negative populations was monitored. e, Cell cycle distribution of A549 cells pulsed with EdU, then cultured with or without 200 µM G for the indicated time. Percentage of total cells that are EdU-positive and in G1, S or G2/M phase is shown. f, mVenus-Gem1 fluorescent reporter to assess cell cycle dynamics in live cells. g, Representative images from live-cell imaging of A549 cells expressing mVenus-Gem1 cultured with or without 200 µM G (see also Supplementary Videos 1–3). h, Fraction of cells cultured with or without 200 µM G that began the experiment in G1 phase and entered S phase (assessed by live-cell imaging of A549 cells expressing mVenus-Gem1; 76 cells were analysed). i, Duration of S/G2 phase in cells cultured with or without 200 µM G (assessed by live-cell imaging of A549 cells expressing mVenus-Gem1; 115 cells were analysed). j, Cell cycle distribution of A549 cells synchronized in G2 phase with 4.5 µM RO-3306 for 18 h, then released from arrest and treated with standard culture media (untreated), 25 µM G (low G) or 200 µM G (high G) as indicated. k, dNTP levels in A549 cells 21 h after release from RO-3306 and subsequent treatment with or without low G or high G as indicated. dNTP levels in unsynchronized cells cultured with or without low G or high G for 24 h are also shown. dNTPs were measured using LCMS. Data are presented as mean ± s.d. of three biological replicates. Source numerical data are available in source data.

Source data