Extended Data Fig. 5. Imbalanced nucleotides induce replication stress signaling.
a, Western blot assessing phosphorylation of Chk1 and Chk2 in A549 cells cultured for the indicated time in standard media (Untreated) or media lacking leucine, or with addition of 200 µM guanine or 20 µM deoxyguanosine, with or without addition of 200 µM adenine as indicated. Levels of vinculin are also shown as a loading control. b, Western blot assessing phosphorylation of Chk1 and Chk2 in 143B cells cultured in standard conditions (Untreated) or treated with 200 µM guanine (G), 1 µM lometrexol (LTX), 1 µM brequinar (BRQ), 1 mM thymidine (T), 2.5 mM adenine (A), or 1.5 mM deoxyadenosine (dA) for the indicated time, or treated with 1 µM Torin for 24 hours. Levels of vinculin are also shown as a loading control. c, Western blot assessing phosphorylation of Chk1 and Chk2 in H1299 cells cultured in standard conditions (Untreated), treated with 200 µM G, 1 mM T, 2.5 mM A, or 1.5 mM dA for the indicated time, treated with 1 µM Torin for 24 hours, or treated with 1 µM LTX or 1 µM BRQ for 96 hours. Levels of vinculin are also shown as a loading control. d, Western blot showing levels of p53 and p21 in A549 cells in standard media (Untreated) or cultured for the indicated time with the indicated concentration of G. Levels of vinculin are also shown as a loading control. e, Comet assay to assess the presence of both single-stranded DNA and double-stranded DNA breaks (DNA damage) in A549 cells treated without (Untreated) or with 200 µM guanine for 24 hours. 529 cells were analyzed. Data are presented as mean + /- SD. Numerical source data and unprocessed blots are available in source data.