Fig 2: Generation of forebrain neuron-specific FT knockout mice. A Schematic representation of the 3-step breeding strategy to generate APP/PS1 mice harboring homozygous FT knockout in forebrain neurons (APP/PS1/FTnKO) and littermate controls. B Representative immunoblot images of FT-β and HDJ-2, a well-known farnesylated protein that is widely used as a marker for farnesylation, in cortical, hippocampal, cerebellar tissue homogenates of WT and FTnKO mice at 3 and 6 months of age. Unfarnesylated HDJ-2 band is detected in the forebrain region (cortex and hippocampus), but not in the cerebellum, of FTnKO mice at 3 months, and the level is elevated at 6 months, confirming both brain region- and age-dependent deletion of FT. C, D Immunoblot analysis of representative small GTPases in membrane (M) and cytosolic (C) fractions of cortical tissue lysates of FTnKO mice compared with WT controls (n = 5/genotype). Membrane-associated FT substrates, farnesylated H-Ras and Rheb, are reduced significantly or trending reduction in FTnKO compared with WT controls, whereas membrane levels of GGT substrate RhoA are not affected, confirming the specificity of FT deletion. E Representative immunoblot images of HDJ-2 in cortical tissue homogenates of APP/PS1 and APP/PS1/FTnKO mice at 9–10 months of age. F, G Immunoblot analysis of representative small GTPases in membrane (M) and cytosolic (C) fractions of cortical tissue lysates from APP/PS1 and APP/PS1/FTnKO mice at 9–10 months of age (n = 5/genotype). Wilcoxon signed rank-order test, 2-tailed; #p = 0.078; *p < 0.05; **p < 0.01

Fig 3: TET activates autophagy by inhibiting Rheb/mTORC1 signaling. (A–D) Effect of TET on mTOR activation. Immunoblot (A) and quantitative analysis (B–D) showed the expression level of mTOR phosphorylation (p-mTOR), mTOR downstream substrates (p-P70, p-4E-BP1). (E,F) Representative images of immunofluorescence monitored by confocal microscopy (J) and quantitative analysis (K) showed the expression of p-4E-BP1. Green staining is p-4E-BP1, blue staining indicates nuclei. Scale bars: 20 μm. (G–I) Effect of mTOR activation on autophagy regulation. Primary MLFs were cotreated with TET (4 μM) and MHY1485 (5 μM) for 24 h. Immunoblot (G) and quantitative analysis (H–I) showed the expression of autophagy markers (MAP1LC3-I/II, SQSTM1), mTOR activation (mTOR, p-mTOR). (J,K) Effect of TET on the expression of Rheb protein. Representative immunoblot analysis (J) and quantitative analysis (K) show the expression of Rheb protein. (L) Representative of qPCR analysis showed the mRNA expression of Rheb. (M) Effect of TET on Rheb activation. Co-immunoprecipitation was performed using agarose beads conjugated to an antibody directed against the GTPγS and GDP. Immunoblot analysis was performed to check for protein expression of activated Rheb (Rheb/GTP), total Rheb. Total Rheb includes the inactive GDP-Bound form (GDP) and activated Rheb (Rheb/GTP). Values in bar graph are presented as means ± SEM (n = 5). Two-way ANOVA with Tukey’s multiple comparison test (n = 5). *p < 0.05.

Fig 4: Effects of PYP15 on the mTORC1 downstream signaling components in DEX-stimulated C2C12 myotubes. C2C12 myotubes were treated for 24 h with 100 μM DEX in the absence or presence of 500 ng/mL PYP15. (A) The protein levels for Rheb, p-p70S6K, p70S6K, p-S6, and S6 were assessed as described in Section 4. (B) The protein levels for p-4EBP1, 4E-BP1, and eIF4E were assessed as described in Section 4. GAPDH was the loading control. The values are the mean ± SD of three independent experiments. * P < 0.05 vs. corresponding control; # P < 0.05 vs. corresponding only DEX treatment.

Fig 5: TET improves pulmonary fibrosis by activating autophagy through Rheb/mTOR in MLFs. Primary MLFs were treated with TGF-β1 (0 or 10 ng/ml) for 24 h in the presence/absence of TET (4 μM), 3 MA (500 nM), RAP (50 nM), or MHY1485 (a small molecule mTOR activator, 5 μM). (A,B) Representative immunoblot analysis (A) and quantitative analysis (B) showed the expression of fibrosis-associated proteins. (C,D) The effect of autophagy on TGF-β1-induced primary MLFs. Immunoblot (C) and quantitative analysis (D) showed the expression of ECM deposition, myofibroblasts transdifferentiation markers. (E,F) Confocal microscopy images (E) and relative fluorescence intensity of α-SMA (F). α-SMA was visualized in green, DAPI-stained nuclei in blue. Scale bars: 50 μm. (G) MLFs transfected with nonspecific nonsilencing negative siRNA control (si-NC) or ATG7 small interfering RNA (si-ATG7) for 48 h were treated with TET and TGF-β1. Representative immunoblot analysis showed the expression of fibrosis-associated proteins. (H) Cells transfected with the empty vector plasmid (vector) or Rheb overexpression plasmid (Rheb) for 48 h and then incubated with TET and TGF-β1. Immunoblot of fibrosis-associated proteins. (I) TET-attenuated fibrosis was associated with mTOR activation. Immunoblot showed the expression of fibrosis-associated proteins. Values in bar graph are presented as means ± SEM (n = 5). Two-way ANOVA with Tukey’s multiple comparison test (n = 5). *p < 0.05.