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

Figure 6.

Figure 6

Nuciferine disrupts Rag GTPases–Ragulator interaction and inhibits mTORC1 activity. (A) Representative immunoblotting of Raptor, TFEB, p-TFEB-Ser211, ULK1, p-ULK1-Ser757, S6K, p-S6K-Thr389, S6, p-S6-Ser240/244, 4E-BP1 and p-4E-BP1-Ser65 in HEK293T cells and quantification of p-TFEB-Ser211/TFEB, 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 (transfection with lentiviruses expressing Flag-Raptor in the presence of AA) group. HEK293T cells were transfected with lentiviruses expressing Flag-Raptor or Flag-Raptor-RHEB15, then transfected with GFP-TFEB and starved of AA for 1 h in the presence or absence of 100 μmol/L nuciferine. (B–D) HepG2 cells were transfected with lentiviruses expressing Flag-Raptor or Flag-Raptor-RHEB15 and maintained in medium containing 2% bovine serum albumin and treated with 400 μmol/L palmitic acid (PA) for 12 h, then treated with or without 100 μmol/L nuciferine for 12 h. (B) The triglyceride content in HepG2 cells. (C) Representative images of Oil red O (original magnification 40 ×) staining in HepG2 cells. (D) Representative immunoblotting of IR, p-IR, AKT and p-AKT in HepG2 cells and quantification of p-IR/IR, p-AKT/AKT and expressed as fold change relative to control (transfection with lentiviruses expressing Flag-Raptor and treatment with PA and insulin) group. Cells were treated with 100 nmol/L insulin for 30 min before they were harvested. (E) Representative Co-IP analysis to assay interactions between HA-RagC and TFEB, mTOR and P18 in HepG2 cells. HepG2 cells were transfected with HA-RagC and treated with 100 μmol/L nuciferine for 12 h or starved of AA for 1 h. (F) Representative images of RagC 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 TFEB, RagA, RagC, mTOR, P18 and LAMP1 in lysosomes from liver tissues and quantification of TFEB/LAMP1, RagA/LAMP1, RagC/LAMP1, mTOR/LAMP1, P18/LAMP1 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.