Fig 2: NIM811 induced a transient upregulation of UPR and autophagy. (a and b) In all, 0 µM NIM811 transiently induced UPR signaling (P-EIF2a, Bip, ATF4, CHOP upregulation) at 6 h, but except for Bip, the activation of UPR mediators disappeared at 9 h. Tunicamycin (Tm) and thapsigargin (Tg) were used as positive controls for the UPR. (c) In total, 10 µM NIM811 was added to GFP-LC3B transfected U251 cells and imaged by fluorescence microscopy at 0 h and 6 h. Scale bar=25 µm. LC3 puncta were detected at 3–6 h. LC3 puncta formation occurred before the appearance of vacuoles. (d) LC3-I and -II conversion occurred at 4–6 h of 10 µM NIM811 treatment accompanied by p62 degradation. (e and f) In all, 10 µM NIM811 incubation caused p62 accumulation at 24 h, which persisted at later time points (40–46 h). (g) Immunofluorescence staining of SQSTM1/p62 (red) and nucleus (blue) after 24 h vehicle (left) or 10 µM NIM811-treated (right) U251 cells. Scale bar=25 µm

Fig 3: LARP1 in suppression of 5'TOP mRNA translation by CDK1i. (A and B) Polysome association of 5'TOP (RPLP0 and PABPC4) and ORF size-matched non-TOP (EIF2S1 and NCL) mRNAs was analyzed by polysome fractionation and subsequent qPCR analysis from DMSO- or Ro3306-treated (10 µM, 4 h) HeLa cells. (C and D) Polysome association of 5'TOP (RPS6 and RPS7) and ORF size-matched non-TOP (EIF2S1, CDKN1A) mRNAs was analyzed by polysome fractionation and subsequent qPCR analysis from DMSO- or Ro3306-treated (10 µM, 4 h) HEK293T WT, LARP1-/-, and LARP1-/- + LARP1 cells. (E) Polysome profiles were recorded from HEK293T WT and LARP1-/- cells treated with either DMSO or Ro3306 (10 µM) for 4 h. The percentage of polysomal ribosomes is represented in the inset (mean ± SEM, n = 3), and the graph on the right side depicts the fold change in polysomal ribosomes (4 h Ro3306/DMSO control). (F) HeLa cells were treated with DMSO or Ro3306 (10 µM) for 4 h, and cell lysates were prepared for RNA-IP using anti-LARP1 or IgG control antibody. The amount of LARP1- and IgG-bound 5'TOP (RPL29 and RPL32) and non-TOP ß-actin mRNAs was determined by qPCR (mean ± SEM, n = 3). Protein levels of LARP1 and GAPDH (negative control) in input and IP samples were measured by Western blot analysis. In E and F, statistical significance was determined by paired one-tailed Student’s t test.

Fig 4: IDO-mediated anoxia-induced apoptosis and reoxygenation-induced ferroptosis molecular pathways. IDO-mediated anoxia-induced apoptotic molecular pathway is depicted on the left. IDO-mediated reoxygenation-induced ferroptotic molecular pathway is depicted on the right. AIMP3/p18, aminoacyl-tRNA synthetase-interacting multifunctional protein-3/p18; AhR, aryl-hydrocarbon receptor; ATF3, activating transcription factor 3; ATF4, activating transcription factor 4; ATM/ATR, ataxia-telangiectasia mutated/ataxia-telangiectasia and Rad3 related protein complex; CHOP, C/EBP homologous protein; CC3, cleaved caspase-3; CYP1A1, cytochrome P450 family 1 subfamily A polypeptide 1; DR5, death receptor 5; IDO, indoleamine 2,3-dioxygenase 1; Kyn, kynurenine; p-, phosphorylated; eIF2a, eukaryotic translation initiation factor-2a; GCN2K, general control nonderepressible-2 kinase; MRS, methionyl-tRNA synthetase; p53, p53; ROS, reactive oxygen species; Trp, tryptophan.

Fig 5: Overexpression of CMT-GARS mutants, E71G, G240R and ?ETAQ, induces integrated stress response (ISR) via phosphorylation of eIF2a. (A) Expression of CMT-GARS mutants induces phosphorylation of eIF2a. HEK293T cells were transfected with plasmids encoding WT, E71G, G240R, ?ETAQ GARS or an empty vector. For a positive control of eIF2a phosphorylation, cells were treated with thapsigargin. Cell lysates were analyzed by western blotting using antibodies against phosphorylated eIF2a (P-eIF2a), eIF2a (loading control), and myc (GARS-myc), as indicated on the left. (B) Schematic representation of reporter constructs used in transfection experiments: RL and FL are the same as in Figure 2A. ATF4-RL carries 5'UTR of ATF4 gene. (C) E71G, G240R and ?ETAQ GARS mutants activate expression of ATF4 reporters. Human HEK293T cells were co-transfected with plasmids encoding one of the Renilla luciferase reporters (RL, ATF4-RL), FL and myc-tagged GARS, either WT or indicated mutant. As additional controls, empty vector and NSP1-encoding plasmids were used instead of GARS plasmid. RL activity was normalized to that of FL and presented as a percentage of luciferase activity produced in the presence of empty vector for each Renilla reporter. Values represent means ± SD from three experiments. (D) Puromycylation assay shows that CMT-GARS-mediated translational repression is preserved upon inhibition of eIF2a phosphorylation. HEK293T cells were transfected with plasmids encoding WT, E71G, G240R or ?ETAQ GARS and inhibitor of eIF2a phosphorylation phosphorylation GCN2-IN-1 was added were indicated. After puromycin treatment, cells were lyzed and lysates were analyzed by western blotting with antibodies against phosphorylated eIF2a (P-eIF2a), eIF2a (loading control), myc (GARS-myc) and puromycin, as indicated on the left. PAAG stained with coomassie is provided to show total protein loading.