Figure 5. Glutamine depletion inhibits cell proliferation and decreases intracellular ATP levels. (A) ATP levels were reduced by glutamine depletion. WT and atg5^−/− MEFs were seeded in complete medium to reach 50% confluence on the day of treatment, and then grown in serum-supplemented DMEM (without glutamine) in the presence or absence of glutamine (4 mM), 2-DG (10 mM) or methylpyruvate (10 mM) for the indicated time periods prior to harvesting. ATP assays were performed as described in Materials and Methods. Cell proliferation was inhibited by glutamine depletion and 2-DG treatment. WT and atg5^−/− MEFs were seeded in complete medium to reach 10% confluence on the day of treatment, and then grown in serum-supplemented DMEM in the presence and absence of glutamine (4 mM) or in the full medium with 2-DG (10 mM) for indicated time periods. Relative cell proliferation (determined by the cell proliferation assay described in methods) for each treatment was calculated by comparing to that at 0 h, set as 1. Results from three independent experiments are shown as mean ± SD. The Student’s t-test was performed to determine the difference between (a) control (24/48 h) and -Gln (24/48 h); (b) control (24/48 h) and 2-DG (24/48 h). (B) Both DM-2-KG and NAC rescued cell proliferation. WT and atg5^−/− MEFs were seeded in complete medium to reach 10% confluence on the day of treatment, and then grown in glutamine-depleted DMEM with DM-2-KG (7 mM), NAC (2 mM), or both. Cell proliferation was assessed by cell proliferation assay. Significance was calculated using the Student’s t-test between: WT MEFs in Gln(-) (24/48/72 h) and WT MEFs in DM-2-KG/NAC/combination of DM-2-KG and NAC (24/48/72 h); atg5^−/− MEFs in Gln(-) (24/48/72 h) and atg5^−/− MEFs in DM-2-KG/NAC/combination of DM-2-KG and NAC (24/48/72 h); WT MEFs in NAC (24 h) and atg5^−/− MEFs in NAC (24 h). (C) Supplementing with NAC rescues the proliferation of glutamine-deprived cells. Real-time cell proliferation was monitored using an RTCA DP Analyzer. The m5-7 cells were maintained in medium without or with Dox to turn off Atg5 expression (right panel). Glutamine was withdrawn in the presence and absence of NAC supplements and cell growth determined and recorded every 30 min. (D) Minimal glutamine concentration required to support the proliferation of WT and atg5^−/− MEFs. Cell proliferation was determined as described in Material and Methods. Briefly, 5000 cells were seeded into 96-well plate over night and incubated overnight prior to switching to the medium with indicated glutamine concentration (0, 0.5 and 4 mM), while 4 mM representing the complete medium. Cell viability was determined at 24, 48 and 72 h afterwards and the % relative cell proliferation was calculated as that of 72 h as 100%. Gln(-), grown in DMEM without glutamine; Gln(+), grown in DMEM with glutamine; DM-2-KG, dimethyl-2-ketoglutarate; 2-DG, 2-deoxyglucose; NAC, N-acetyl-cysteine; Dox, doxycycline. (A and B) Results are shown as mean ± SD from three independent experiments. *p < 0.05.