Glutamine deficiency specifically triggers DNA damage accumulation independent of cell death.

<p>(A) MEF and PC3 cells were cultured in complete (Cont) or glutamine free medium (No Gln) for 24 hours. Cells were fixed for immunofluorescence with γH2AX antibody and DAPI. Scale bar 100 μm. Data represent mean ± SD of 4 independent cell cultures, *** <i>P</i> < 0.001, shown is the percentage of cells showing >10 foci. (B) The indicated cancer cell lines were cultured in complete or glutamine-free medium. (C) MEF cells were cultured in medium deprived of the indicated amino acid for 48 hours. (D) EB3 and MEF cells were cultured in complete medium, glutamine-free or glucose-free medium for 6 hours. Early apoptotic cell death was determined by AnnexinV staining with flow cytometry. (E) MEF cells were cultured in either low glutamine (0.1 mM) or low glucose (1 mM) for the indicated time points; relative cell growth was determined by CellTiter-Glo assay. (B-E) The treated cells were lysed for immunoblotting using indicated antibodies. (F) <i>Bak</i>^-/- /<i>Bax</i> ^-/- MEF cells were starved of glutamine for 48 hours, and cells were lysed for immunoblotting. (G) MEF and PC3 cells were cultured in complete or glutamine-free medium overnight. PC3 cells were also treated with 3.4 μM Doxo for 4 hours to induce DNA DSB. The cells were fixed for immunofluorescence using the indicated antibodies. Scale bar 10 μm. (H) EB3 cells were cultured in complete or glutamine-free medium overnight; cells were lysed for western blot analysis using the indicated antibodies. Arg, arginine; Asn, asparagine; Asp, aspartate; Cont, control; Doxo, doxorubicin; DSB, double strand breaks; EAA, essential amino acids; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; Glc, glucose; Gln, glutamine; Gly, glycine; Leu, leucine; MEF, mouse embryonic fibroblast; Nam, Namalwa; p-ATM, ataxia-telangiectasia mutated; p-Chk1, checkpoint kinase 1; Ser, serine.