Fig 2: TRF1 regulation by PI3K and AKT inhibitors. a Structurally diverse PI3K and PI3K/mTOR inhibitors used in the study. b PI3K and mTOR data generated internally and reported in literature. c Representative western blot images of phosphorylated AKT-Ser473 and total AKT in CHA-9.3 mouse lung tumor cell line at 24 h after treatment with PI3K, AKT, and mTOR inhibitors as indicated. d Percent inhibition of TRF1 foci by immunofluorescence and of AKT phosphorylation at S473 (pAKT) in CHA-9.3 mouse lung tumor cell line at 24 h after treatment with the indicated inhibitors relative to TRF1 levels and to pAKT levels in control cells treated with DMSO. The inhibitors were tested at 10 μM except GSK-2126458 that was used at 1.0 μM (in c and d). Error bars represent standard deviation. The Student’s t test was used for statistical analysis; P values are shown. n number of independent experiments

Fig 3: ATF4 is a direct phosphorylation target of mTOR in response to mitochondrial stress.a ATF4 phosphorylation recognized by a context-dependent (S*P) phosphorylation-specific antibody is increased by Rheb co-expression and inhibited by Torin1. HEK293T cells transfected with the indicated plasmids were immunoprecipitated (IP) with anti-Flag antibody and analyzed by western blot analysis. When applicable, Torin1 (250 nM) were added 2 h before harvest. TCL total cell lysate. b mTOR directly phosphorylates ATF4 in vitro. In vitro kinase assay was performed with recombinant GST-tagged human mTOR purified from baculovirus-infected insect cells and recombinant His-tagged human ATF4 with or without Torin1 (250 nM). Arrows indicate the mobility shifts likely separating the hyperphosphorylated and nonphosphorylated ATF4. c The ratios of the phosphorylated and nonphosphorylated peptides containing the phosphorylation sites of ATF4 from a kinase assay performed similar to (b), as determined by mass spectrometry. d The identified mTOR-targeted phosphorylation sites on ATF4 with the vertebrate orthologs aligned below, with numbering according to the amino-acid sequence of human ATF4 protein. NTD N-terminal domain, BD Basic domain, CLZ C-leucine zipper. The highly conserved putative TOR signaling (TOS) motif was also highlighted. e Validation of the two commercially available antibodies that specifically recognize ATF4 phosphorylation at Ser166 and Thr173, respectively. HEK293T cells transfected with the indicated plasmids were immunoprecipitated with anti-Flag antibody and analyzed by western blot assay. Torin1 (250 nM) was added 2 h before harvest. f Increased ATF4 Ser166 and Thr173 phosphorylation upon Dox treatment, which was inhibited by ConA and Torin1. Wild-type MEFs were pretreated with DMSO, ConA (200 nM) or Torin1 (250 nM) for 1 h, and then co-treated with or without Dox (30 μg/mL) for 2 h, immunoprecipitated with anti-ATF4 antibody and analyzed by western blot assay. g Increased ATF4 phosphorylation upon mitochondrial, but not ER stress inducers. Wild-type MEFs were with treated with Antimycin A (AntiA, 2 μM), Oligomycin (Olig, 2 μM), or Tunicamycin (TM, 1.5 μg/mL) for 2 h, immunoprecipitated with anti-ATF4 antibody and analyzed by western blot assay. A similar amount of immunoprecipitated ATF4 protein was loaded for different conditions to compare phosphorylation changes in (f, g).

Fig 4: Mitochondrial stress induces v-ATPase-dependent mTORC1 activation at the lysosomal surface.a Western blot analysis showing time-dependent changes of proteins in MEFs pretreated with DMSO or ConA (200 nM) for 1 h, and then co-treated with or without Dox (30 μg/mL) for 0–8 h. b Western blot analysis showing time-dependent changes of proteins in MEFs treated with Antimycin A (AntiA, 2 μM) or Tunicamycin (TM, 1.5 μg/mL) for 0–8 h. c ConA inhibits mitochondrial stress-induced lysosomal localization of mTOR in MEFs. MEFs were pretreated with DMSO control or ConA (200 nM) for 1 h, and then co-treated with or without Dox (30 μg/mL) for 3 h, cells were then fixed and co-stained with mTOR (green) and lysosome marker Lamp1 (red) antibodies. The arrows indicate the mTOR-lysosome co-localized puncta. Scale bar, 10 μm. d, e Western blot analysis (d) and qRT-PCR results (n = 5 mice for each group) (e) of kidney samples from 9–10-week-old male C57BL/6J mice treated with vehicle control (ctrl) or Dox (50 mg/kg) for 24 h. Error bars denote SEM. Statistical analysis was performed by two-tailed unpaired Student’s t test (*P < 0.05; **P < 0.01; ***P < 0.001; N.S. not significant).