Journal: Cells

Article Title: Ursodeoxycholic Acid Regulates Hepatic Energy Homeostasis and White Adipose Tissue Macrophages Polarization in Leptin-Deficiency Obese Mice

doi: 10.3390/cells8030253

Figure Lengend Snippet: Ursodeoxycholic acid (UDCA) alleviates high free fatty acid (HFFA)-induced hepatocyte lipogenesis, reactive oxygen species (ROS) production, and mitochondrial dysfunction in AML12 cells. AML12 cells were treated with 1 mM HFFA with 10, 30, 100 μM UDCA. ( A ) Lipid accumulation display using Oil Red O stain (red). ROS levels were measured using DCFH-DA (green) stain. Images of AML12 cells stained with Mito Tracker for mitochondria (red). qRT-PCR analysis of ( B ) Complex I, II, III, IV, and V mRNA expression in AML12 cells. Relative mRNA expression was normalized to Gapdh and then normalized to the controls. ( C ) Immunofluorescence analysis of SREBP1c (green), CD36 (red), NF-κB (green), and FXR (green) expression, and DAPI (blue) for nuclear. Scale bar, 25 μm. qRT-PCR analysis of ( D ) Srebp-1c, Fas , and Scd-1 mRNA expression in AML12 cells. In all panels, results are expressed as the mean ± S.E.M. of five independent experiments, and statistical significance of differences between means was assessed using an unpaired Student’s t -test (* p ≤ 0.05; 0 mM HFFA vs. 1 mM HFFA. # p ≤ 0.05; 1 mM HFFA vs. 1 mM HFFA+ 100 μM UDCA). UDCA, ursodeoxycholic acid; HFFA, high free fatty acid; ROS, reactive oxygen species; SREBP-1c, sterol regulatory element-binding protein-1c; CD36, cluster of differentiation 36; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; FXR, farnesoid X receptor; Fas , fatty acid synthase; Scd-1, stearoyl-CoA desaturase-1; qRT-PCR, quantitative real-time polymerase chain reaction; Gapdh , glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Alpha mouse liver 12 (AML12) cell line (passages 7–10, obtained from Bioresource Collection and Research Centre, Taiwan) was grown at 37 °C in Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F-12 (DMEM/F12), 10% fetal bovine serum (FBS), 2 mmol/L l -Glutamine and 100 μg/mL penicillin and streptomycin (both from Gibco, AntiSel, Greece) at 5% CO 2 .

Techniques: Staining, Quantitative RT-PCR, Expressing, Immunofluorescence, Binding Assay, Real-time Polymerase Chain Reaction

Journal: Frontiers in Cell and Developmental Biology

Article Title: TMAO-Activated Hepatocyte-Derived Exosomes Impair Angiogenesis via Repressing CXCR4

doi: 10.3389/fcell.2021.804049

Figure Lengend Snippet: Isolation and characterization of exosomes from hepatocyte culture supernatant. (A) Nanovesicles with diameters around 100 nm were isolated and purified from the AML12 cell culture supernatant, which possessed the characteristic size range of exosomes (Exos) under electron microscopes. Bar: 200 nm. (B) The size distribution of the Exos showed no significant difference between trimethylamine-N-oxide (TMAO)-free group (Control-Exos) and TMAO-Exos. (C) Exosomal markers of CD9 and TSG101 were enriched in Exos groups, and the negative markers of calnexin were detected only in whole cell lysate. (D) TMAO was undetectable in Control-Exos, but a small quantity of TMAO remained in TMAO-Exos. Data were expressed as mean ± standard error of the mean (SEM). n = 3, independent t -test was performed for comparisons; ** p < 0.01 versus Control-Exos. (E) Exosomes were labelled with DiI and co-cultured with human aortic endothelial cells (HAECs) for 24 h, and it was shown that DiI-labeled Exos could be taken up by cells (×400 magnification).

Article Snippet: AML12 cells (iCell Bioscience Inc, Shanghai, China) were cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) containing Exos-depleted serum (ViVaCell, Shanghai, China), and treated with TMAO (Tokyo Chemical Industry, Japan) at a physiological concentration of 50 μmol/l for 48 h. Exos were isolated and purified from the culture supernatant using differential centrifugation and then resuspended in phosphate-buffered saline (PBS), as described in our previous study ( ).

Techniques: Isolation, Purification, Cell Culture, Control, Labeling