Extended Data Fig. 6: ATR signaling impacts the fate of cells with imbalanced nucleotides.
From: Nucleotide imbalance decouples cell growth from cell proliferation
a, Schematic outlining how ATR and ATM kinases respond to replication stress and DNA damage. The ATR and ATM targets Chk1 and Chk2 activate downstream effectors that halt cell cycle progression. AZ20 is an inhibitor of ATR kinase activity. b, Western blot assessing phosphorylation of Chk1 and Chk2 in A549 cells cultured in standard media (Untreated) or treated for the indicated time with 200 µM guanine (G) with or without 50 nM AZ20. Levels of vinculin are also shown as a control. c, Proliferation rates of 143B and H1299 cells treated with the indicated concentration of G with or without 50 nM AZ20 (ATRi). d, Cell death measured in A549 cells cultured for 96 hours in standard conditions (Untreated) or treated with 200 µM G with or without 50 nM ATRi as indicated. e, Proliferation rates of H1299 cells cultured in standard conditions (none) or treated with 2 mM adenine (A), 1.5 mM deoxyadenosine (dA), 200 µM G, or 1 mM thymidine (T), with or without 50 nM ATRi as indicated. f. Proliferation rates (left) and Western blot assessing phosphorylation of Chk1 (right) in A549 cells treated with or without 200 µM G with or without 0.6 µM of the ATR inhibitor VE821. Levels of vinculin are also shown as a control. g, Proliferation rates of A549 cells cultured in standard conditions (Untreated) or treated with 1 µM lometrexol (LTX) or 1 µM brequinar (BRQ) with or without 50 nM AZ20 (ATRi) as indicated. h, Cell fate as assessed using live-cell imaging of A549 mother cells expressing the mVenus-Gem1 reporter that were in S/G2 phase at the time of addition of 200 µM G with or without 50 nM AZ20 (ATRi). The fate of mother cells in S/G2 not exposed to excess G is also shown (Untreated). 83 cells were analyzed. i, Cell fate as assessed using live-cell imaging of A549 daughter cells expressing the mVenus-Gem1 reporter that were born after the addition of 200 µM G with or without 50 nM AZ20 (ATRi). The fate of daughter cells not exposed to excess G is also shown (Untreated). 158 cells were analyzed. j. SA-β-galactosidase activity was assayed in A549 cells cultured in standard conditions (Untreated), treated with 200 µM G for 96 hrs, or treated with 200 µM G with or without 50 nM AZ20 (ATRi) for 96 hrs and then switched to untreated media for 7 days (Recovery). Cells treated with Palbociclib for 7 days were included as a control. k, Mean volume of A549 cells measured over time after release from treatment with G with or without AZ20 (ATRi) as described in Fig. 5h. l, Cell cycle distribution as assessed by DNA content of A549 cells treated with 200 µM G with or without 50 nM AZ20 (ATRi) for the indicated time. Data are presented as mean ± SD of 3 biological replicates. Source numerical data and unprocessed blots are available in source data.
