Fig 2: Rapamycin induces autophagy in time-dependent and concentration-dependent manners in iPSCs. iPSCs were maintained in six-well plates and medium was renewed daily. Images of LC3B staining were taken at 4 days of rapamycin treatment at 0, 10, 100, or 200 nM respectively (a–d) and for 200 nM of rapamycin treatment at 1, 3, and 6 days respectively (e–h), showing concentration-dependent and time-dependent induction. (i) Proteins were extracted from iPSCs treated with rapamycin at 0, 1, 10, 100, 200, or 300 nM for 4 days, and immunoblotted with antibodies against LC3B, phosphorylated ULK1, phosphorylated p70S6K, or ß-actin. (j–l) Relative abundance of the LC3B-II/I ratios (j), p-p70S6K (k), and p-ULK1 (l) was quantified using ImageJ software against a loading control ß-actin. Data were presented as mean ± SEM, *p < 0.05, **p <0.01. Bar = 10 µm a–h

Fig 3: The ISR and mTORC1 are active in livers of mice treated with HF. WT and whole body Gcn2–/– mice were injected with HF (0.5 mg/kg) or 0.5% DMSO vehicle. Following 8 h, one group of mice were sacrificed and liver tissues were measured for ISR and mTORC1 signaling, charging of tRNAPro, and histological analyses. Additionally, another group of mice received a second injection of HF at 24 hours and were sacrificed after 44 h for liver analyses. (A) The indicated proteins were measured by immunoblot analyses with specific antibodies. Bar graphs to the right of the immunoblot panels show quantification of phosphorylated S6K1 and eIF2a from three biological replicates. The bar graphs are represented as the means ± SD and *P < 0.05. (B) Measurements of the percent uncharged tRNAPro (top panel) and relative total tRNAPro (bottom panel) by RT-qPCR. Significant differences between treatments are indicated. *P < 0.05; **P < 0.005; ***P < 0.0005; ****P < 0.0001. (C) Left panel: hepatic PRODH expression was measured by immunoblot analyses with indicated antibodies; right panel: quantification of hepatic PRODH expression. (D) The ratio of hepatic glutamate to proline amino acid was measured. (E) Representative images of H&E staining of hepatic tissue for lipid droplets accumulation. Images were captured using light microscopy (100× magnification) (left panel). Quantitation is featured in the accompanying bar graph (right panel). Significant differences between treatments are indicated. ***P < 0.0008; ****P < 0.0001. (F) Hepatic tissue levels of TG were measured in lipid extracts. (G) Liver ALT levels were measured in the serum samples. Bar graphs are represented as the means ± SD, and significant differences between treatments are indicated: *P < 0.05; **P < 0.005.

Fig 4: SHP2 positively regulate ERK1/2 and AKT signaling pathwaysA. SHP2 overexpression promotes dephosphorylation of pPaxillin(Y118) and pSrc(Y527), as well as ERK1/2, AKT and S6K activation. Consistently, SHP2 knockdown attenuates dephosphorylation of pPaxillin(Y118) and pSrc(Y527), as well as ERK1/2, AKT and S6K activation under B. non-stimulation or C. HGF stimulation.

Fig 5: C1-Ten PTPase activates mTORC1. (a) Effect of nephrin Y1138F mutant on IRS-1-mediated mTORC1 activation. HEK293 cells were transfected with GFP vector, GFP nephrin WT, or GFP nephrin Y1138F for 24 h. The cells were serum-starved for 18 h, then treated with 10 nM insulin for 30 min. mTORC1 activation was measured by immunoblotting of phospho- and total- S6K1. Data are means ± SEM (n = 3). (b) Effect of C1-Ten overexpression on mTORC1 activation. Human podocytes were transduced with Adenovirus (Ad) GFP, Ad C1-Ten WT or Ad C1-Ten CS for 48 h. Protein expression of phospho- and total-S6K1 were detected by Western blotting. Data are means ± SEM (n = 3). (c) Effect of C1-Ten knockdown on the HG-mediated mTORC1 activation. mTORC1 activation was presented by immunoblotting of phospho- and total- S6K1. Data are means ± SEM (n = 3). *P <0.05.