Figure 5. 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).