Figure S1. DS has no cytotoxicity in HepG2 cells. HepG2 cells were cultured in high-glucose DMEM containing 10% FBS at 37°C in 5% CO₂, seeded on a 96-well plate (5,000 cells/well), and incubated with control (0.1% DMSO) and DS (0, 1.25, 2.5, 5, 10, 25, 50, or 100 μM) for 24 h. Then, the cells were incubated with MTT (10 μL of 5 mg/mL, Sigma–Aldrich) for 3 h at 37°C before DMSO (200 μL) was added. The absorbance was measured on a Synergy H4 microplate reader (Biotek, Vermont, USA) at 570 nm, and the background absorbance was measured at 630 nm. The background absorbance was subtracted from the signal absorbance to obtain normalized absorbance values. The colorimetric signal obtained was proportional to the cell number. Data are statistically analyzed as the means ± SEM (n = 5).

Figure S2. The mRNA expression of FXR and TGR5 is elevated by overexpressing the relevant plasmids. HEK293T cells were transfected with overexpression plasmid of phFXR or phTGR5 for 48 h. Then, the cells were subjected to mRNA expression determination to verify the transfection efficiency. Relative mRNA expression of FXR (A) and TGR5 (B). n = 3. All data are statistically analyzed as the means ± SEM. *p < 0.05 versus Control.

Figure S3. DS has no effect on activating the transactivity of PPARγ and LXRα, β. HEK293T cells were cotransfected with pCMXGal-hPPARγ, LXRα, and β, and the Gal4 reporter vector and MH100 × 4-TK-Luc were cotransfected into HEK293T cells for 24 h followed by DS (50 μM) and rosiglitazone or T0901317 (20 μM) treatment for 24 h. Then, the cells were subjected to luciferase activity determination. The data showed that DS had no influence on the transactivation of PPARγ and LXRα, β. Transient transfection reporter assay of PPARγ (A), LXRα (B), and LXR β (C). n = 3. All data are statistically analyzed as the means ± SEM. *p < 0.05 versus Control.

Figure S4. FXR knockdown blocks DS-induced SHP expression. HepG2 cells were transfected with FXR and control siRNA for 48 h or 24 h followed by treatment with control (0.1% DMSO) and DS (50 μM) for another 24 h. Total RNA from the cells was extracted using a TRIzol reagent. First-strand cDNA was synthesized with a cDNA Synthesis Kit (Fermentas, Madison, WI). The relative gene expression of FXR and SHP were analyzed on an ABI StepOnePlus real-time PCR system (Applied Biosystems, USA) using the 2-^ΔΔCt method. The data indicated that FXR silencing strikingly decreased the expression of FXR (A) and blocked the upregulation of SHP induced by DS (B). All gene expression was normalized using β-actin as an internal control. Data are statistically analyzed as the means ± SEM (n = 3). *p < 0.05 versus Control; NS: no significance.

Figure S5. Central DS treatment increases mouse energy expenditure. DIO C57BL/6 mice were implanted stereotaxically with guide cannulas (26 gauge, 5 mm, RWD Life, China) into the right lateral ventricle at 0.3 mm anterior–posterior, 1.0 mm midline-right, and 2.5 mm dorsal–ventral from bregma by a stereotaxic apparatus as previously described (RWD Life, China). After a 7-day recovery, the mice were tested for the success of surgery with angiotensin (Tocris Bioscience, UK). Overnight-fasted surgery mice were randomly divided into two groups, receiving 1-week i.c.v. injection of saline or DS (40 μg/2 μL/mouse) by guide cannula. Then, the mice were housed in metabolic cages to measure metabolic parameters, including oxygen consumption (VO₂), carbon dioxide generation (VCO₂), and respiratory exchange ratio (RER), by using the Columbus Instruments Comprehensive Lab Animal Monitoring System (CLAMS) to evaluate the beneficial effect of i.c.v. infusion of DS on improving energy metabolism. The results showed that DS inhibited VCO₂ and RER in DIO mice. (A, D) Oxygen consumption (VO₂). (B, E) Carbon dioxide generation (VCO₂). (C, F) Respiratory exchange ratio (RER). All data are statistically analyzed as the means ± SEM (n = 3). *p < 0.05, saline versus deoxyschizandrin group.

Table S1. Sequences of the human primers used in real-time PCR.

Table S2. Sequences of the mouse primers used in real-time PCR.