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. 2021 Dec 22;12(6):2869–2886. doi: 10.1016/j.apsb.2021.12.012

Figure 5.

Figure 5

Nuciferine inhibits mTORC1 activity and decreases TFEB phosphorylation. (A) Representative co-immunoprecipitation (Co-IP) analysis to assay interactions between HA-TFEB and RagA, RagC, mTOR and 14-3-3 in HepG2 cells. HepG2 cells were transfected with HA-TFEB and treated with 100 μmol/L nuciferine for 12 h or starved of amino acids (AA) for 1 h. (B) Representative immunoblotting of p-TFEB-Ser211, GFP-TFEB, p70 S6 kinase (S6K), p-S6K-Thr389, ribosomal protein S6 (S6), p-S6-Ser240/244, EIF4E-binding protein 1 (4E-BP1) and p-4E-BP1-Ser65 in HEK293T cells and quantification of p-TFEB-Ser211/TFEB, p-S6K-Thr389/S6K, p-S6-Ser240/244/S6, p-4E-BP1-Ser65/4E-BP1 and expressed as fold change relative to control (no treatment) group. HEK293T cells were transfected with GFP-TFEB, and then treated with different concentrations of nuciferine (0, 10, 25, 50, 100 or 200 μmol/L) for 12 h. (C) Representative immunoblotting of HA-RagC, TFEB, unc-51-like kinase 1 (ULK1), p-ULK1-Ser757, S6K, p-S6K-Thr389, S6, p-S6-Ser240/244, 4E-BP1 and p-4E-BP1-Ser65 in HepG2 cells and quantification of p-ULK1-Ser757/ULK1, p-S6K-Thr389/S6K, p-S6-Ser240/244/S6, p-4E-BP1-Ser65/4E-BP1 and expressed as fold change relative to control (no treatment) group. HepG2 cells were transfected with or without active RagC (S75L), and then treated with 100 μmol/L nuciferine for 12 h or 250 nmol/L Torin1 for 1 h. (D) HEK293T cells were analyzed by GFP-TFEB fluorescence and quantified to calculate the percentage of cells showing TFEB unclear localization. Scale bar, 20 μm. HEK293T cells were transfected with GFP-TFEB, and then treated with 100 μmol/L nuciferine for 12 h or 250 nmol/L Torin1 for 1 h. (E) HepG2 cells were analyzed by immunofluorescence and quantified to calculate the percentage of cells showing TFEB unclear localization. Scale bar, 20 μm. Cells were transfected with RagC or active RagC (S75L), and then treated with 100 μmol/L nuciferine for 12 h or 250 nmol/L Torin1 for 1 h. (F) Representative images of mTOR immunofluorescence staining and LysoTracker in HepG2 cells and quantification of colocalization coefficient. HepG2 cells were starved of AA for 1 h in the presence or absence of 100 μmol/L nuciferine. Scale bar, 10 μm. (G) Representative immunoblotting of S6K, p-S6K-Thr389, S6, p-S6-Ser240/244, 4E-BP1 and p-4E-BP1-Ser65 in the livers from different groups and quantification of p-S6K-Thr389/S6K, p-S6-Ser240/244/S6, p-4E-BP1-Ser65/4E-BP1 and expressed as fold change relative to ND group. Mice were treated as described in Fig. 1. n = 6 mice per group were used to analyze the results. All experiments were repeated at least 3 times. Data were expressed as the mean ± SEM; ∗P < 0.05, ∗∗P < 0.01.