Fig 2: Proposed model elucidating the effect of ULK1 loss on SFA-induced lipotoxicity in cells. Our study suggests that besides the impairment of autophagy-mediated signaling, ULK1 downregulation in a lipotoxic environment leads to increased activation of RPS6KB1. This in turn causes increased nuclear localization of NCOR1 in the nucleus. NCOR1-mediated inactivation of NR1H/LXRs causes suppression of Scd1 transcription hence leading to an increased SFA/MUFA ratio. This would further decrease the generation of neutral LDs and eventually cause cell death via lipotoxicity.

Fig 3: Branched-chain amino acids (BCAAs) and leucine (Leu) decrease cisplatin-mediated autophagy and increase cisplatin sensitivity.A, B Western blot analysis of the protein levels of BCAT1, mTOR, p70S6K, 4E-BP1 and their phosphorylated counterparts and LC3-I/II in Hela cells treated with cisplatin (20 μM) or in combination with BCAAs (5 mM) or Leu (2 mM) for 24 h. C, D Hela cells were treated with increasing concentrations of cisplatin alone or in combination with BCAAs (5 mM) or Leu (2 mM) for 24 h. Cell viability was assessed by CCK-8 assays. E Representative image of Hela cells transfected with pCMV-mCherry-GFP-LC3B. The cells were treated with cisplatin (20 μM) or Leu (2 mM) alone or together for 24 h. Scale bars: 25 μm. Three independent experiments were performed. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 4: The knockdown of EZH2 induced the increase of trophoblast autophagy and mTOR pathway inhibition under hypoxic conditions. (A) Significant decreases in EZH2, mTOR and S6K1 mRNA levels were tested in JEG-3 and HTR-8 cells transfected with EZH2 siRNA by qRT-PCR. (B,C) The trophoblast cell lines were transfected with a EZH2 siRNA under hypoxic conditions, and BECN1, P62 and LC3 expression levels were tested by western blotting analysis. Empty vector (scramble) cells served as controls. (D,E) Cell proliferation was evaluated by a CCK-8 assay and EdU flow cytometry. The two trophoblast cell lines were transfected with EZH2 siRNA with or without AD-MSC exosomes treatment under hypoxic conditions. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 5: J3 inhibits mTOR pathway. A WT-A549 were treated with 20 μM of J3 or 1 μM of Rap for 6 h followed by immunostaining of ATG16L1 and ATG12. ATG16L1 and ATG12 positive structures were indicated by arrowhead. Scale bar = 20 μm. B ATG16L1 or ATG12 dots from at least 50 cells were counted for quantification of different groups. C WT and ATG4B-deficient HeLa cells were incubated with 20 μM of J3 for 6 h, then ATG4B, LC3-I, LC3-II (in WT-HeLa), and pro-LC3 (in ATG4B-deficient HeLa, with asterisk) were analyzed by immunoblotting. D WT and ATG5-deficient MEF cells were incubated with 20 μM of J3 for 6 h, then ATG5, LC3-I, and LC3-II were analyzed by immunoblotting. E WT and ATG16L1-deficient HeLa cells were incubated with 20 μM of J3 for 6 h, then ATG16L1, LC3-I, and LC3-II were analyzed by immunoblotting. F WT- and ATG5KO-MEF cells stably expressing GFP-LC3 were treated by 20 μM of J3 for 6 h. The distribution of GFP-LC3 dots was analyzed. G WT-HeLa cells were treated with 20 μM of J3 or 1 μM of Rap for 6 h. The total protein level and the phosphorylation level of mTOR, ULK1, and S6K were analyzed by immunoblotting as indicated. H Cells were treated as G, then relative phosphorylation level of each protein was calculated. Data are presented as mean ± sem from three individual experiments. *p < 0.05, ***p < 0.001, ns means not significant