Fig 2: MnCl2 maintains mTORC1 activity during starvation in human cells.(A–B) Human U2OS (A) and HEK293T (B) cells were starved for all amino acids and supplemented with increasing concentrations of MnCl2 as indicated for 2 hr. Phosphorylation of the mTORC1 downstream targets RPS6KB (ribosomal protein S6 kinase B) and RPS6 (ribosomal protein S6) was assessed by immunoblot analysis. A control with cells incubated in the presence of all proteinogenic amino acids (+AA) was included as a positive control. (C) Human U2OS were starved for all amino acids (-AA) and supplemented with 0.35 mM of MnCl2 for 2 hr. Phosphorylation of RPS6KB, RPS6, EIF4EBP1, ULK1, and AKT1 was assessed by immunoblot analysis. A control with cells incubated in the presence of all proteinogenic amino acids (+AA) was included as a positive control. (D–E) Human U2OS cells were treated as in (C) for 4 hr either in the absence (D) or the presence (E) of chloroquine (CQ). Autophagic marker MAP1LC3I/II was then analyzed by immunoblot. (F–G) GFP-MAP1LC3 expressing U2OS cells were incubated as in (D). GFP-MAP1LC3 aggregation was assessed by confocal microscopy (F) and quantified using ImageJ software (G). Scale bar indicates 10 µm. Values were normalized to -AA condition. Graphs represent mean ± SD, with n=3 (*p<0.05, ANOVA followed by Tukey’s test). (H) Model of Mn2+-driven TORC1 activation. The Smf1/2 natural resistance-associated macrophage protein (NRAMP) transporter-dependent increase in cytoplasmatic Mn2+ levels favors TORC1-Mn2+ binding and ATP coordination, leading to TORC1 hyperactivation. NRAMP transporters are part of a feedback control mechanism impinged by TPRC1 (dashed lines). Figure 5—source data 1.Quantification of microscopy images for graph shown in Figure 5G. Figure 5—source data 2.Uncropped blots shown in Figure 5A–E. Figure 5—source data 3.Raw blots shown in Figure 5A–E.

Fig 3: TME conditions regulate the phosphorylation levels of p70S6K, rpS6 and eIF2a. HUVECs or Ea.hy926 were exposed to normoxic control (Ctrl), TME (1% O2, 1% FBS, 2.5 mM glucose, 7.5 mM lactate and pH 6.5) or hypoxic (Hyp; 1% O2) conditions as indicated, followed by lysis and western blotting. NHE1 was inhibited by cariporide (10 µM) or knocked down by siRNA-treatment where indicated. a Representative western blots of p-Thr389p70S6K and total p70S6K and quantification of p-p70S6K protein levels normalized to total p70S6K and relative to the untreated control condition for 24 h for HUVEC. GAPDH is shown as a loading control. b, c Representative western blots of p-Ser235/236rpS6 and quantifications of p-rpS6 protein levels relative to the untreated control condition for 24 h for HUVEC (b) and Ea.hy926 (c). GAPDH and p150 are shown as loading controls. d, e Representative western blots of p-Ser51eIF2a and quantifications of p-eIF2a protein levels relative to the untreated control condition for 24 h for HUVEC (d) and Ea.hy926 (e). p150 is shown as loading control. Data are presented as means with SEM error bars, with n = 5 except Hyp without/with cariporide for which n = 3. *, ** and *** indicates p < 0.05, p < 0.01 and p < 0.001, respectively, as obtained by two-way ANOVA with Bonferroni’s multiple comparison post-test. The two-way ANOVA test revealed a significant difference in p-p70S6K/p70S6K (p < 0.0001), p-rpS6 (p < 0.0001 for HUVEC and p < 0.05 for Ea.hy926) and p-eIF2a (p < 0.05 for HUVEC and p < 0.01 for Ea.hy926) between conditions (Ctrl, TME, Hyp). Also, p-p70S6K/p70S6K significantly changed with cariporide (p < 0.01)

Fig 4: Global Changes in the Translatome upon Acute ER Stress(A) Scatterplot of fold changes (Tm 2 h versus 0 h) as determined by anota2seq using total or polysome-associated mRNAs. Transcripts with changes in translational efficiency are shown in orange (up) or purple (down).(B) Same as (A), but for Tm 4 h versus 0 h.(C) Rps6 and Mrps16 mRNA abundances across the polysomal fractions at 2 and 4 h following Tm injection quantified by qPCR.(D) Same as (C), with Asns and Pah mRNAs at 2 h and Nedd4 and Cul4b mRNAs at 4 h following Tm injection.In (C) and (D), representative results of three independent experiments are shown; data are mean ± SEM (n = 3).(E) Boxplot of translation activities as determined by anota2seq of mRNAs downregulated at 2 h following Tm. The group of proteins synthetized at the ER were defined by Jan et al. (2014).(F) Same as (E) but at 4 h following Tm.(G) Boxplot of translation activities as determined by anota2seq of all mRNAs downregulated (Down), all mRNAs downregulated excluding the mRNAs encoding for the RPs (Down w/o RP), cytosolic RP mRNAs (CytoRP), and mitochondrial RP mRNAs (MitoRP), respectively, at 2 h following Tm.(H) Same as (G), but 4 h following Tm.(I) Density plot of translational activities of mRNAs downregulated at 2 h following Tm and represented at 0, 2, and 4 h following Tm.(J) Same as (I), but with translational activities of downregulated mRNAs at 2 h represented at 0 and 10 h following Tm.***p < 0.001, as determined by an unpaired Wilcoxon test. See also Figures S2–S5.

Fig 5: Translationally Downregulated Proteins following eIF2a Phosphorylation Are Long-Lived(A) Experimental design (top panel) to measure mitochondrial translation. 35S methionine/cysteine incorporation into newly translated mitochondrial proteins (encoded by mtDNA) following chloramphenicol (Cm; 50 µg/mL) or Tm (2.5 µg/mL) treatment of HEK293T cells for the indicated times. Cytoplasmic translation was inhibited using emetine (100 µg/mL). ATF4 was used as a positive control for the Tm treatment. Representative results of at least three independent experiments are shown.(B) Representative immunoblots showing the abundance of RPL4, RPS6, MRPL44, MRPL28, ATF4, and tubulin proteins at the indicated times after treatment of HeLa cells with 2.5 µg/mL Tm. ATF4 was used as a positive control for the Tm treatment.(C) Same as (B), but following 20 µg/mL cycloheximide (CHX) treatment. POMP (a short-lived protein) was used as a control for the CHX treatment.(D) Histogram showing the distribution of protein half-lives encoded by translationally down- or upregulated mRNAs (Down or Up) at 2 h following Tm injection.(E) Boxplot of half-lives of proteins encoded by the translationally down- or upregulated mRNAs (Down or Up), all 79 CytoRPs, or all 82 MitoRPs at 2 h following Tm injection.(F) Same as (E), but with exclusion of the RPs (Down w/o RPs).In (E) and (F), protein half-lives were analyzed from Sandoval et al. (2013). **p < 0.01 and ***p < 0.001, as determined by an unpaired Wilcoxon test. See also Figures S8 and S9.