Research Paper Volume 13, Issue 17 pp 20962—20991

Figure 8. Nucleosides and NEAA restore escape from TIS in Gln-deprived conditions. (A) Schematic overview of glutamine usage in cancer cells. Gln glutamine, Glu glutamate, Asn asparagine, GSH glutathione, α-KG alpha-ketoglutaric acid, NEAA non-essential amino acids, GLS glutaminase, GS Glutamine synthetase. (B) Doxorubicin-induced senescent A549 cells were grown with (+Gln) or without (−Gln) glutamine in medium containing 0.1 mM each adenosine, guanosine, cytidine, thymidine, uridine, or in combination (Mix, 0.1 mM each). Colonies that evaded the senescent growth arrest were stained and counted. The data shown here represent three experiments exhibiting similar effects. A representative image of the colony escape assay is shown. (C) Doxorubicin-induced senescent A549 cells were grown with (+Gln), or without (−Gln) glutamine plus NEAA, in the presence or in the absence of 2 mM MSO. Colonies that evaded the senescent growth arrest were stained and counted. The data shown here represent three experiments exhibiting similar effects. A representative image of the colony escape assay is shown. (D) Expression of GS and SLC1A5 proteins was analyzed in parental A549 cells, grown with (+) or without (−) glutamine or without glutamine plus NEAA (NEAA) for 72 hours. Filters were stripped and reprobed with anti-β-tubulin antibodies as a loading control. GS and SLC1A5 levels, normalized to the relative β-tubulin levels, are reported as fold change of Gln-supplemented sample.