Fig. 4: Atf4 uORF1 is required for s-ISR.

a,l, Representative western blot of the indicated proteins in mouse ES cells expressing control (shCon), eIF2Bε or eIF2Bε + eIF4E shRNAs (a) or in MEFs treated with glucose (0–25 mM, 16 h) (l). LiCl (10 mM) or Torin 1 (250 nM) was used for the specified final hours of the 16 h treatment with glucose-free medium (l) (n = 3 independent experiments). b, Schematic of the Atf4 uORF1 start codon mutation in mouse ES cells. c, Representative western blot of the indicated proteins in WT or ΔuORF1 mouse ES cells treated with vehicle or Tg (400 nM) for the specified times (left and middle) or expressing control or Eif2b5 shRNAs (right). (n = 3 independent experiments). d,e, OCR (d) and ECAR (e) of WT and ΔuORF1 mouse ES cells. Oligomycin (Oligo), FCCP, rotenone (Rot) and antimycin A (AA), and monensin (Mon) were injected as indicated. P values, two-tailed Student’s t-test. Data are presented as mean ± s.e.m. (n = 5 independent experiments). f,g, Quantification of ATP production from oxidative phosphorylation (J ATP ox) or glycolysis (J ATP glyc) (f) and fold change in bioenergetic capacity (g) in WT versus ΔuORF1 mouse ES cells. P values, two-tailed Student’s t-test. Data are presented as mean ± s.e.m. (n = 5 independent experiments). h, J ATP glyc and J ATP ox values in WT and ΔuORF1 mouse ES cells for basal (square), FCCP (triangle) or monensin (circle) conditions. Maximum theoretical boundaries for J ATP ox (horizontal line) and J ATP glyc (vertical line) are indicated. P values, two-tailed Student’s t-test. Data represent mean ± s.e.m. (n = 5 independent experiments). i, Schematic of [¹³C]glutamine tracing in the CAC. PEP m + 3 and asparagine m + 4 are labelled by carbons derived from [¹³C]glutamine. CS, citrate synthase. j,k, Fractional enrichment (j) and relative ion abundance (k) of PEP m + 3 in ΔuORF1 cells versus WT. P values, two-tailed Student’s t-test. Data represent mean ± s.e.m. (n = 3 independent experiments).

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