Additional file 1 of Didymin alleviates metabolic dysfunction-associated fatty liver disease (MAFLD) via the stimulation of Sirt1-mediated lipophagy and mitochondrial biogenesis
Creators: name: Yang, Jing-wen
name: Zou, Ying
name: Chen, Jun
name: Cui, Chen
name: Song, Jia
name: Yang, Meng-meng
name: Gao, Jing
name: Hu, Hui-qing
name: Xia, Long-qing
name: Wang, Li-ming
name: Lv, Xiao-yu
name: Chen, Li
name: Hou, Xin-guo
Publisher: name: figshare
Year: 2023
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Pharmacology
subject: Chemical Sciences not elsewhere classified
subject: Biological Sciences not elsewhere classified
subject: Cancer
subject: Computational Biology

description: Additional file 1: Figure S1. Didymin promotes the expression of Sirt1 by activating FoxO3a. (A) Western blot analysis of FoxO3a proteins in AML12 cells after siRNA transfection (n=3). (B) Western blot analysis of Sirt1 and FoxO3a proteins in AML12 cells after siRNA transfection (n=3). Data are expressed as mean ± SD. *P &lt; 0.05, **P &lt; 0.01. Figure S2. Heatmap of lipophagy-related genes. The red boxes indicate proteins directly regulated by FoxO3a, histone H4 K16, and TFEB. Figure S3. Didymin suppresses apoptosis by restoring lipophagy in PA-treated AML12 cells. (A) Representative images of TUNEL staining in AML12 cells (Scale bar = 100 μm). Quantification of the percentage of TUNEL-positive cells (n=3). (B) Apoptosis analysis of AML12 cells by flow cytometry. The results of quantitative analyses of apoptosis rate (n=3). (C) Western blot analysis of Bax, Bcl2, cleaved-caspase3, and caspase3 proteins in AML12 cells (n = 3). Data are expressed as mean ± SD. *P &lt; 0.05, ***P &lt; 0.001 PA vs. PA+Didymin. # P &lt; 0.05, ## P &lt; 0.01, ### P &lt; 0.001, #### P &lt; 0.0001 control vs. PA. Figure S4. Liver morphology and statistical analysis of western blot results. (A) Liver morphology (B) Statistical analysis of western blot results in Figure 7A (n=3). (C) Statistical analysis of western blot results in Figure 7B (n=3). (D) Statistical analysis of western blot results in Figure 7E (n=3). (E) Statistical analysis of western blot results in Figure 7F (n=3). Data are expressed as mean ± SD. *P &lt; 0.05, **P &lt; 0.01 MAFLD vs. MAFLD+Didymin. # P &lt; 0.05, ## P &lt; 0.01, ### P &lt; 0.001, #### P &lt; 0.0001 control vs. MAFLD. Figure S5. Western blot analysis of PGC-1α and FoxO3a in hepatocytes (n=3). Data are expressed as mean ± SD. *P &lt; 0.05, **P &lt; 0.01 MAFLD vs. MAFLD+Didymin. # P &lt; 0.05, ## P &lt; 0.01 control vs. MAFLD. Figure S6. Sirt1-in-1 inhibits the activation of SIRT1 by Didymin in AML12 cells. (A) TG contents in AML12 cells (n=4). (B) Western blot analysis of NRF1 and TFAM in hepatocytes (n=3). (C) Western blot analysis of Beclin1, p62, and LAMP1 in hepatocytes (n=3). Data are expressed as mean ± SD. **P &lt; 0.01, ***P &lt; 0.001, ****P&lt; 0.0001 PA vs. PA+Didymin. ## P &lt; 0.01, ### P &lt; 0.001, #### P &lt; 0.0001 control vs. PA. Figure S7. Cell viability of AML12 cells treated with PA and different concentrations of Didymin (n=4). Data are expressed as mean ± SD. ****P&lt; 0.0001 PA vs. PA+Didymin.