Fig. 5: An essential role of ATF4 and its phosphorylation by mTORC1 in UPR^mt activation.

a qRT-PCR results (n = 4 biologically independent samples) of wild-type (WT) and Atf4^−/− MEFs treated with or without Dox (30 μg/mL) or Antimycin A (AntiA, 2 μM) for 24 h. b The OCR of WT or Atf4^−/− MEFs at basal or after sequential addition of Oligomycin (Olig), FCCP and AntiA/Rotenone. The basal and maximum OCR was statistically analyzed (n = 6 biologically independent samples). c Atf4 knockout leads to disrupted mitochondrial network upon mitochondrial stress. MitoTracker staining of WT or Atf4^−/− MEFs treated with or without Dox (30 μg/mL) or AntiA (2 μM) for 24 h. The average mitochondrial network perimeter and area were analyzed by ImageJ with a Mito-Morphology macro (n = 3 independent experiments). Scale bar, 10 μm. d Western blot analysis of Atf4^−/− MEFs stably expressing empty vector (vector), the wild-type ATF4 (WT-ATF4), the phospho-defective mutant (5A-ATF4), and an ATF4 mutant carrying a point mutation of the bulky phenylalanine residue 94 in the TOS motif to alanine (F94A-ATF4). e qRT-PCR results (n = 4 biologically independent samples) of Atf4^−/− MEFs stably expressing vector, wild-type, 5A or F94A forms of ATF4, treated with or without Dox (30 μg/mL) for 24 h. Error bars denote SEM. Statistical analysis was performed by two-tailed unpaired Student’s t test in (b), or by ANOVA followed by Tukey post hoc test in (a, c, e) (**P < 0.01; ***P < 0.001; N.S. not significant).