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: 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

Journal: Journal of Clinical and Translational Research

Article Title: IL-23 and IL-17A are not involved in hepatic/ischemia reperfusion injury in mouse and man

doi:

Figure Lengend Snippet: A: Nitrite production after 24-h incubation of RAW 264.7 cells with AML12 hepatocyte-derived DAMPs. Data are plotted as mean ± SEM with N = 7-8 per group. B: Real-time ROS production after 24-h incubation of RAW 264.7 cells with AML12-derived DAMPs. Data are plotted as mean ± SEM with N = 4 per group. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001 compared to the medium group. Abbreviations: c, centrifuged; LPS, lipopolysaccharide; PMA, phorbol 12-myristate 13-acetate; DCF, dichlorofluorescein.

Article Snippet: The mouse hepatocyte cell line AML12 was cultured in William’s E (WE) medium (Lonza, Basel, Switzerland) supplemented with 10% (v/v) fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, MA), penicillin (100 U/mL, Thermo Fisher Scientific), streptomycin (100 μg/mL, Thermo Fisher Scientific), insulin (5 μg/mL, Sigma-Aldrich, St. Louis, MO), L-glutamine (2 mM, Thermo Fisher Scientific), and hydrocortisone hemisuccinate (50 μM, Sigma-Aldrich).

Techniques: Incubation, Derivative Assay

Journal: Journal of Clinical and Translational Research

Article Title: IL-23 and IL-17A are not involved in hepatic/ischemia reperfusion injury in mouse and man

doi:

Figure Lengend Snippet: Pro-inflammatory signaling by DAMP-exposed RAW 264.7 macrophages. A: TNFα, B: IL-1β, and C: IL-6 mRNA expression after 1-h and 6-h DAMP incubation. All results are presented as fold upregulation compared to medium incubation (N = 3-4 per group). D-G: Luciferase reporter assay of RAW 264.7 NF-κB/LUCPorter cells following medium-, DAMP-, or LPS stimulation after D: 6 h, E: 12 h, and F: 24 h (N = 3 per group). G: Luciferase reporter assay after stimulation with DAMPs derived from ischemia-subjected necrotic cells, medium, or LPS after 24 h of exposure (N = 3 per group). Luciferase activity is expressed as the fold change relative to control. H: IL-23 production by murine macrophages in response to AML12 hepatocyte-derived DAMPs measured by ELISA in RAW 264.7 cell supernatant and corrected for protein (N = 4 per group). All data represent mean ± SEM. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001 compared to the medium samples.

Article Snippet: The mouse hepatocyte cell line AML12 was cultured in William’s E (WE) medium (Lonza, Basel, Switzerland) supplemented with 10% (v/v) fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, MA), penicillin (100 U/mL, Thermo Fisher Scientific), streptomycin (100 μg/mL, Thermo Fisher Scientific), insulin (5 μg/mL, Sigma-Aldrich, St. Louis, MO), L-glutamine (2 mM, Thermo Fisher Scientific), and hydrocortisone hemisuccinate (50 μM, Sigma-Aldrich).

Techniques: Expressing, Incubation, Luciferase, Reporter Assay, Derivative Assay, Activity Assay, Control, Enzyme-linked Immunosorbent Assay