Fig. 6. ATR signalling promotes dNTP availability during unperturbed S phases.
a, Cell cycle distribution of A549 cells corresponding to western blots in b. Cells were treated with 9 µM RO-3306 for 18 h to arrest cells in G2 phase, then released for the indicated time. b, Phosphorylation of Chk1 and Chk2 in A549 cells treated with 9 µM RO-3306 for 18 h to arrest cells in G2 phase, then released for the indicated time. Levels of vinculin are also shown. c, Cell fate assessed using live-cell imaging of A549 mother cells expressing mVenus-Gem1 that were in G1 phase at the time of 50 nM AZ20 (ATRi) addition. The fate of mother cells in G1 not exposed to ATRi is also shown (untreated). Fifty-five cells were analysed. d, Cell fate of A549 daughter cells expressing the mVenus-Gem1 reporter born to mother cells either in S/G2 phase (left) or G1 phase (right) at the time of 50 nM ATRi addition. Mother cells that were in S/G2 phase when ATRi was added went through a partial S phase with ATR inhibited, whereas mother cells that were in G1 phase went through a full S phase with ATR inhibited. The fate of daughter cells not exposed to ATRi is also shown (untreated). For left and right graphs, 104 and 107 cells were analysed, respectively. e, dNTP levels in A549 cells synchronized in G2 phase by treating with 4.5 µM RO-3306 for 18 h, then released from RO-3306 and treated with DMSO or 50 nM ATRi for the indicated times. Unsynchronized cells (unsync.) were treated with DMSO or 50 nM ATRi for 24 h as indicated. f, NTP levels in A549 cells synchronized in G2 phase by treating with 4.5 µM RO-3306 for 18 h, then released and treated with DMSO or 50 nM ATRi for the indicated times. Unsync. cells were treated with DMSO or 50 nM ATRi for 24 h as indicated. Nucleotide levels were measured using LCMS. Data are presented as mean ± s.d. of three biological replicates. Source numerical data and unprocessed blots are available in source data.