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: Heliyon

Article Title: Oral administration of kynurenic acid delays the onset of type 2 diabetes in Goto-Kakizaki rats

doi: 10.1016/j.heliyon.2023.e17733

Figure Lengend Snippet: KYNA enhances mRNA and protein expression of hepatic UCP genes. (A, B) Real-time quantitative PCR analysis (n = 6) of gene expression in HepG2 cells after 48 h of 10 μmol/L (K10) or 100 μmol/L (K100) KYNA treatment compared with control group (white punctuated line). (C, D) Western blot analysis of UCP2 expression in HepG2 cells after 48 h of 100 μmol/L KYNA treatment (n = 3), (E, F) Real-time quantitative PCR analysis of gene expression in HepG2 cells after 2 h treatment with 10 μmol/L (K10) or 100 μmol/L (K100) KYNA (n = 6), (G) Quantitative PCR analysis of gene expression in AML-12 cells after 48 h treatment with 10 μmol/L (K10) or 100 μmol/L (K100) KYNA (n = 6), (H) Expression of hepatic energy metabolism genes in GK rats after oral administration 25 mg/kg KYNA (KYNA) or NaCl (Ctrl) (I) Comprehensive analysis of transcriptomic pathways in KYNA-treated HepG2 cells after 48 h KYNA, 100 μmol/L (n = 3). Statistical analysis using t -test, * p < 0.05.

Article Snippet: HepG2 human liver cells and AML-12 mouse liver cells (Procell, China) were cultured in MEM basal medium (Gibco, China) containing 10% fetal bovine plasma (Gibco, Australia).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: PLoS ONE

Article Title: miR-146a Ameliorates Liver Ischemia/Reperfusion Injury by Suppressing IRAK1 and TRAF6

doi: 10.1371/journal.pone.0101530

Figure Lengend Snippet: (A) Left panel. Representive FACS of hepatocellular apoptosis. Right panel. The percent of apoptotic cells were measured in three separate experiments. (B) The ROS levels were analyzed using DHE red fluorescence probe in AML12 cells stimulated with 0 (a), 10 ng/ml (b), 20 ng/ml (c), 40 ng/ml (d) TNF-α for 6 h. (C) Immunoblotting of phosphorylated and total JNK. Results are mean ± SD. Results are mean ± SD. * , P<0.05 and ** ,P<0.01, *** ,P<0.001 versus negative control.

Article Snippet: About 5×10 5 AML12 cells were stimulated with 0, 10, 20, 40 ng/ml TNF-α (Peprotech, NJ, USA) for 6 h. To determine the ROS level, treated cells were washed with serum-free DMEM/F12 culture medium and incubated with 5 μM dihydroethidium (DHE, Beyotime, Nantong, China) at 37°C for 30 min. Then images were observed using a fluorescent inverted microscope (Nikon, Tokyo, Japan).

Techniques: Fluorescence, Western Blot, Negative Control

Journal: Journal of Clinical and Translational Hepatology

Article Title: PCSK9 and APOA4 : The Dynamic Duo in TMAO-induced Cholesterol Metabolism and Cholelithiasis

doi: 10.14218/JCTH.2024.00403

Figure Lengend Snippet: (A–E) APOA4 , PCSK9 , HMGCR, ABCG5 , and ABCG8 mRNA expression levels in TMAO-treated and control AML12 cells (n = 3 per group). (F) Immunofluorescence staining for APOA4, PCSK9, HMGCR, ABCG5, and ABCG8 in cells from each group. ** p < 0.01, *** p < 0.001. TMAO, trimethylamine-N-oxide; APOA4, apolipoprotein A4; PCSK9, proprotein convertase subtilisin/kexin type 9; ABCG5, ATP-binding cassette sub-family G member 5; ABCG8, ATP-binding cassette sub-family G member 8; HMGCR, 3-hydroxy-3-methylglutaryl-CoA reductase.

Article Snippet: The murine normal hepatocyte line AML12 was acquired from Cyagen Biosciences Inc. (Shanghai, China) and cultured using AML12 cell-specific culture medium (Procell, Wuhan).

Techniques: Expressing, Control, Immunofluorescence, Staining, Binding Assay