Extended Data Fig. 7: Amino acid levels remain relatively constant during unperturbed cell cycles and are not impacted by loss of ATR signaling.
From: Nucleotide imbalance decouples cell growth from cell proliferation
a, Cell cycle distribution of A549 cells corresponding to Western blots shown in Fig. 6b. Cells were arrested in G2 phase by treating with 9 µM RO-3306 for 18 hours, then RO-3306 was removed to release from cell cycle arrest for the indicated time. A549 cells in standard culture are also shown (Unsynchronized). Cells were pulsed with EdU for 30 minutes prior to each time point and analyzed as outlined in Fig. 3a. b, Duration of G1 phase and S/G2 phases in A549 cells expressing the mVenus-Gem1 reporter cultured in standard conditions (Untreated) or with 50 nM AZ20 (ATRi), as assessed using live-cell imaging. 87 cells were analyzed. c, Cell fate as assessed using live-cell imaging of A549 mother cells expressing the mVenus-Gem1 reporter that were in S/G2 phase when 50 nM ATRi was added. The fate of mother cells in S/G2 not exposed to ATRi is also shown (Untreated). 55 cells were analyzed. d, Cell cycle distribution of A549 cells corresponding to the metabolite measurements shown in panel e, and in Fig. 6e and 6f. Cells were arrested in G2 phase by treating with 4.5 µM RO-3306 for 18 hours, then RO-3306 was removed to release from cell cycle arrest for the indicated time. Cells were pulsed with EdU for 30 minutes prior to each time point and analyzed as outlined in Fig. 3a. Unsynchronized cells were treated with DMSO or 50 nM ATRi for 24 hours as indicated. e, Levels of the indicated amino acids in A549 cells synchronized in G2 phase by treating with 4.5 µM RO-3306 for 18 hours, then released into the cell cycle for the indicated time. At the time of release from RO-3306, cells were either treated with DMSO or 50 nM ATRi as indicated. Unsynchronized cells (Unsync.) were treated with DMSO or 50 nM ATRi for 24 hours as indicated. All metabolite levels were measured by LCMS. Data are presented as mean ± SD of 3 biological replicates. Source numerical data are available in source data.
