Fig 1: Glutamine‐restrictive therapy fails to enhance the sensitivity of the persister cancer cells. A) Sensitivity test to glutamine restriction in NCI‐H460, NCI‐H1299, NCI‐H1975, A549, PC‐9, NCI‐H661, and HCC827 cells. B) Clonogenic survival assay of NCI‐H460 and NCI‐H1299 cells under 4 mM to 0 mM glutamine conditions. Four thousand cells were cultured in six‐well plates for 10 days, followed by crystal violet staining and imaging. C) Seahorse XF assay measuring the OCR in NCI‐H460 and NCI‐H1299 cells under 4 mM or 0.25 mM glutamine conditions (n = 3 independent experiments). D) Glutamine uptake rate determination in NCI‐H460 and NCI‐H1299 cells cultured for 36 h in 4 mM or 0.25 mM glutamine using a glutamine assay kit (ab197011, Abcam) (n = 3 independent experiments). E) Analysis of differential metabolites between 4 mM and 0.25 mM glutamine conditions on H460 cells. The x‐axis represents groups, and the y‐axis represents expression (n = 3 independent experiments). F) CCK‐8 assay‐based analysis on the effect of glutamine (4 mM or 0.25 mM) on cisplatin treatment sensitivity in NCI‐H460 and NCI‐H1299 persister cells (n = 3 independent experiments). G) Generation of persister cells: NCI‐H460 and NCI‐H1299 cells treated with 4 mM or 0.25 mM glutamine for 1–5 days were switched to complete RPMI‐1640 medium for 2 h to create persister cells. Live cells were sorted, reseeded, and tested for cisplatin IC50. Then, live cells were harvested using the Dead Cell Removal kit (Miltenyi Biotec) (n = 3 independent experiments). Glutamine concentrations of 4 mM to 0 mM are represented by 4Q to 0Q, respectively. Data are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data analyzed using two‐tailed Student's t‐tests (D, E, G) in GraphPad Prism 9.5.0.