Fig. 5: Replication stress signalling promotes cell survival and recovery from nucleotide imbalance.
From: Nucleotide imbalance decouples cell growth from cell proliferation
a, ATR and ATM kinases respond to replication stress and DNA damage. ATR and ATM phosphorylate Chk1 and Chk2, respectively. b, Phosphorylation of Chk1 and Chk2 in A549 cells treated for the indicated time with the indicated concentration of guanine (G). c, Phosphorylation of Chk1 and Chk2 in A549 cells treated for the indicated amount of time with 1 mM thymidine (T), 2.5 mM adenine (A) or 1.5 mM deoxyadenosine (dA). d, Phosphorylation of Chk1 and Chk2 in A549 cells treated for the indicated time with 200 µM G, 1 µM LTX or 1 µM BRQ. Levels of vinculin are also shown in all western blots as a loading control. e, Proliferation rates of A549 cells treated with the indicated concentration of guanine with or without 50 nM of the ATR kinase inhibitor AZ20 (ATRi) as indicated. f, Proliferation rates of A549 cells cultured with or without 2 mM A, 1.5 mM dA, 200 µM G or 1 mM T, with or without 50 nM ATRi as indicated. g, Cell fate of A549 cells expressing the mVenus-Gem1 reporter that were in G1 phase at the time of addition of 200 µM G with or without 50 nM ATRi, as assessed using live-cell imaging. The fate of cells in that were in G1 at the beginning of the experiment and were not exposed to excess G is also shown (untreated). In total, 124 cells were analysed. h, Approach to assess how cells recover from treatment with excess G. Cells were cultured in medium containing 200 µM G with or without 50 nM ATRi for 4 days. Medium was then changed to untreated medium or medium containing 50 nM ATRi, and cell number was determined every 24 h for 14 days thereafter. i, A549 cell number over time after release from treatment with G with or without ATRi treatment as outlined in h. Data are presented as mean ± s.d. of three biological replicates. Source numerical data and unprocessed blots are available in source data.
