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. 2014 Jul 21;206(2):173–182. doi: 10.1083/jcb.201403009

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© 2014 Chen et al.

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Figure 4. — Glutamine starvation induces expression of ATF4, which controls the expression of SLC7A5. (A) CFP-LC3 NRK cells were treated with DMEM in the absence or presence of glutamine or serum. Proteins from cell lysates were analyzed by Western blotting for ATF4 and β-actin. (B) CFP-LC3 NRK cells were pretreated with DMEM for 4 h, and then glutamine or serum was added. Proteins from cell lysates were analyzed for ATF4 and β-actin. (C) CFP-LC3 NRK cells and stable ATF4 knockdown cells were treated with DMEM for the indicated times. Proteins from cell lysates were analyzed for SLC7A5 and β-actin. (D) CFP-LC3 NRK cells and stable ATF4 knockdown cells were treated with DMEM, and C¹³-Leucine was added to the cells at the indicated times. After 10 min of incubation, cells were extracted and the absolute levels of labeled l-Leucine were quantified using LC/MS/MS. (E) CFP-LC3 NRK cells and stable ATF4 knockdown cells were treated with DMEM for the indicated times. The absolute levels of intracellular free amino acids were quantified using LC/MS/MS. Error bars indicate the SD.