Journal: International Journal of Molecular Sciences

Article Title: Novel Benzoxazoles Containing 4-Amino-Butanamide Moiety Inhibited LPS-Induced Inflammation by Modulating IL-6 or IL-1β mRNA Expression

doi: 10.3390/ijms23105331

Figure Lengend Snippet: Compounds 5f and 4d attenuate inflammation in vitro. Effects of compounds (10 µM) on the protein levels of STAT3, IκB, and NF-κB in AML-12 cells. ( a ) Expression levels of inflammation related-proteins in vitro. ( b – d ) Protein expression levels were normal-ized against the indicated protein. * p < 0.05 compared with the group treated with the PBS (vehicle). + p < 0.05 compared with the group treated with the LPS. Data are pre-sented as the mean ± SD.

Article Snippet: The human keratinocytes HaCaT or the alpha mouse liver 12 (AML-12) cells were obtained from Cell Lines Service GmbH (Eppelheim, Germany).

Techniques: In Vitro, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Activation of SIRT1 by Resveratrol Represses Transcription of the Gene for the Cytosolic Form of Phosphoenolpyruvate Carboxykinase (GTP) by Deacetylating Hepatic Nuclear Factor 4? *

doi: 10.1074/jbc.M109.047340

Figure Lengend Snippet: Transcriptional repression of the PEPCK-C gene promoter by SIRT1 activators. HepG2 cells were co-transfected with 200 ng of p2000-Luc plasmid DNA and 50 ng of PKAc plasmid DNA or 50 ng of empty vector DNA, using the protocol described under “Experimental Procedures.” Cells were then treated with the indicated concentrations of SIRT1 activators, namely IsoNAM (panel A), resveratrol (panel B), and fisetin and butein (panel C) for 24 h. The inset in panel A was redrawn from data in Table 5 of Reshef and Hanson (58), in which fed rats were given 5 mmol of nicotinamide (NAM)/kg of body weight for 5 h and the activity of hepatic PEPCK-C determined. A unit of enzyme activity represents the conversion of 1 μmol of substrate to product per min. The IC50 was the concentration of IsoNAM required to achieve 50% inhibition of transcription from the PEPCK-C gene promoter. Values were expressed as mean ± S.E. of triplicate determinations. *, p < 0.001 and **, p < 0.023 as compared with untreated controls. Northern blotting (panel D) and RT-PCR (panel E) were used to determine endogenous mRNA levels of PEPCK-C in FTO-2B and HepG2 cells treated with IsoNAM for various times, or in AML12 mouse hepatocytes treated with IsoNAM at the indicated concentrations. The 18S rRNA stained with ethidium bromide or glyceraldehyde-3-phosphate dehydrogenase mRNA was used as loading controls. Panel F, IsoNAM-induced inhibition of PEPCK-C was reversible. FTO-2B cells were cultured in Krebs-Ringer bicarbonate buffer, supplemented with 2% of bovine serum albumin and 5 mm glucose in the absence (untreated) or presence of 5 mm IsoNAM (treated). After 6 h of treatment, IsoNAM was withdrawn from culture medium. Cells were collected at the time points indicated. PEPCK-C mRNA was measured using RT-PCR; the level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as a control.

Article Snippet: The medium used to culture AML12 cells was also supplemented with insulin-transferrin-selenium (Invitrogen) and 40 ng/ml dexamethasone.

Techniques: Transfection, Plasmid Preparation, Activity Assay, Concentration Assay, Inhibition, Northern Blot, Reverse Transcription Polymerase Chain Reaction, Staining, Cell Culture, Control

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 Immunology

Article Title: Integrated transcriptomic profiling of programmed cell death patterns unveils macrophage-hepatocyte crosstalk via THBS1-CD47 axis in hepatic ischemia-reperfusion injury

doi: 10.3389/fimmu.2026.1769849

Figure Lengend Snippet: OGD/R model induced THBS1 expression in RAW264.7 cells and apoptosis in AML12 cells. (A) Schematic of OGD/R treatment. AML12 cells (lower chamber) and RAW264.7 cells (upper chamber) were co-cultured in serum-free, glucose-free DMEM under hypoxic conditions (1% O2) for 6 hours, followed by re-oxygenation in complete medium for another 3 hours. Control cells were cultured in complete medium under normoxic conditions. (B) THBS1 protein expression in RAW264.7 cells between Control and OGD/R groups. (C) The contents of IL-1β, IL-6, and TNF-α in RAW264.7 cell culture medium between Control and OGD/R groups. (D) The expressions of pro-apoptotic proteins Bax, Cleaved-caspase 3, and anti-apoptotic Bcl2 protein in AML12 cells between Control and OGD/R groups. (E) Cell death in the Control and OGD/R groups was assessed using Hoechst 33342 and PI double staining. Scale bar = 50μm. (F) The mitochondrial membrane potential (ΔψM) of the Control and OGD/R groups was detected using JC-1 staining. Scale bar = 50μm.

Article Snippet: Furthermore, AML12 cells were seeded in 6-well plates 24 hours prior to the establishment of the OGD/R model, with each well receiving 1 μg/mL of recombinant mouse THBS1 protein (MCE, Shanghai, China).

Techniques: Expressing, Cell Culture, Control, Double Staining, Membrane, Staining

Journal: Frontiers in Immunology

Article Title: Integrated transcriptomic profiling of programmed cell death patterns unveils macrophage-hepatocyte crosstalk via THBS1-CD47 axis in hepatic ischemia-reperfusion injury

doi: 10.3389/fimmu.2026.1769849

Figure Lengend Snippet: RAW264.7-specific THBS1 siRNA treatment alleviated OGD/R-induced apoptosis in AML12 cells. (A) Schematic of OGD/R treatment. RAW264.7 cells, separately transfected with siRNA-NC and siRNA-THBS1, were seeded in the upper chamber and co-cultured with AML12 cells (lower chamber) under hypoxic conditions (1% O2) in serum-free, glucose-free DMEM for 6 hours, followed by reoxygenation in complete medium for another 3 hours. (B) THBS1 protein expression in RAW264.7 cells between siRNA-NC and siRNA-THBS1 groups. (C) The contents of IL-1β, IL-6, and TNF-α in RAW264.7 cell culture medium between siRNA-NC and siRNA-THBS1 groups. (D) The expression of pro-apoptotic proteins Bax, Cleaved-caspase 3, and anti-apoptotic Bcl2 protein in AML12 cells between siRNA-NC and siRNA-THBS1 groups. (E) Cell death in the siRNA-NC and siRNA-THBS1 groups was assessed using Hoechst 33342 and PI double staining. Scale bar = 50μm. (F) The mitochondrial membrane potential (ΔψM) of the siRNA-NC and siRNA-THBS1 groups was detected using JC-1 staining. Scale bar = 50μm.

Article Snippet: Furthermore, AML12 cells were seeded in 6-well plates 24 hours prior to the establishment of the OGD/R model, with each well receiving 1 μg/mL of recombinant mouse THBS1 protein (MCE, Shanghai, China).

Techniques: Transfection, Cell Culture, Expressing, Double Staining, Membrane, Staining

Journal: Frontiers in Immunology

Article Title: Integrated transcriptomic profiling of programmed cell death patterns unveils macrophage-hepatocyte crosstalk via THBS1-CD47 axis in hepatic ischemia-reperfusion injury

doi: 10.3389/fimmu.2026.1769849

Figure Lengend Snippet: THBS1 promoted hepatocyte apoptosis by inhibiting PI3K-AKT- NF-κB signaling. (A) The expression of p-AKT, AKT, p-NF-κB p65 and NF-κB p65 proteins in AML12 cells between siRNA-NC and siRNA-THBS1 groups. (B) The expression of pro-apoptotic proteins Bax, Cleaved-caspase 3, and anti-apoptotic Bcl2 protein in AML12 cells among the siRNA-NC, siRNA-THBS1 and siRNA-THBS1+ LY294002 groups. (C) Cell death in the siRNA-NC, siRNA-THBS1 and siRNA-THBS1+ LY294002 groups was assessed using Hoechst 33342 and PI double staining. Scale bar = 50μm. (D) The mitochondrial membrane potential (ΔψM) of the siRNA-NC, siRNA-THBS1 and siRNA-THBS1+ LY294002 was detected using JC-1 staining. Scale bar = 50μm.

Article Snippet: Furthermore, AML12 cells were seeded in 6-well plates 24 hours prior to the establishment of the OGD/R model, with each well receiving 1 μg/mL of recombinant mouse THBS1 protein (MCE, Shanghai, China).

Techniques: Expressing, Double Staining, Membrane, Staining

Journal: Frontiers in Immunology

Article Title: Integrated transcriptomic profiling of programmed cell death patterns unveils macrophage-hepatocyte crosstalk via THBS1-CD47 axis in hepatic ischemia-reperfusion injury

doi: 10.3389/fimmu.2026.1769849

Figure Lengend Snippet: THBS1-mediated hepatocyte apoptosis depended on the CD47 receptor. (A) CD47 protein expression in AML12 cells between Control and OGD/R groups. (B) Schematic of OGD/R treatment. AML12 cells, separately transfected with siRNA-NC and siRNA-CD47, were seeded in the lower chamber and co-cultured with RAW264.7 cells (upper chamber) under hypoxic conditions (1% O2) in serum-free, glucose-free DMEM for 6 hours, followed by reoxygenation in complete medium for another 3 hours. (C) The expression of pro-apoptotic proteins Bax, Cleaved-caspase 3, and anti-apoptotic Bcl2 protein in AML12 cells between siRNA-NC and siRNA-CD47 groups. (D) Cell death in the siRNA-NC and siRNA-CD47 groups was assessed using Hoechst 33342 and PI double staining. Scale bar = 50μm. (E) Schematic of OGD/R treatment. AML12 cells were separately transfected with siRNA-NC and siRNA-CD47, respectively, then cultured in serum-free, glucose-free DMEM supplemented with 1 ng/mL rmTHBS1 under hypoxic conditions (1% O2) for 6 hours, followed by reoxygenation in complete medium for another 3 hours. (F) The expression of pro-apoptotic proteins Bax, Cleaved-caspase 3, and anti-apoptotic Bcl2 protein in AML12 cells among the siRNA-NC, siRNA-NC+ rmTHBS1 and siRNA-CD47+ rmTHBS1 groups. (G) Cell death in the siRNA-NC, siRNA-NC+ rmTHBS1 and siRNA-CD47+ rmTHBS1 groups was assessed using Hoechst 33342 and PI double staining. Scale bar = 50μm. (H) Schematic diagram of the THBS1-CD47 axis’s role in macrophage-hepatocyte crosstalk during HIRI.

Article Snippet: Furthermore, AML12 cells were seeded in 6-well plates 24 hours prior to the establishment of the OGD/R model, with each well receiving 1 μg/mL of recombinant mouse THBS1 protein (MCE, Shanghai, China).

Techniques: Expressing, Control, Transfection, Cell Culture, Double Staining

Journal: Journal of Lipid Research

Article Title: A novel posttranscriptional mechanism for dietary cholesterol-mediated suppression of liver LDL receptor expression

doi: 10.1194/jlr.m049429

Figure Lengend Snippet: Fig. 5. Induction of HNRNPD mRNA expression by cholesterol in cultured hepatic cell lines. HepG2 cells (A) or AML12 cells (B) were seeded in 6-well culture plates and cultured in medium supplemented with 10% LPDS overnight. The next day, DMSO (as vehicle), cholesterol (10 g/ml cholesterol + 1 g/ml 25-hydroxycholesterol), or 5 M RSV was added to the cells for 24 h prior to isolation of total RNA. Hepatic mRNA levels of HNRNPD, LDLR, and GAPDH were assessed by qRT-PCR using specifi c primers with triplicate mea- surement of each cDNA sample. After normalization with GAPDH mRNA levels, the relative levels are presented. The data shown are the summarized results of two separate experiments in which duplicate wells were used in each condition. ** P < 0.01, *** P < 0.001 com- pared with control . CHO, cholesterol. C: HepG2 cells cultured overnight in 10% LPDS medium were treated with RSV, 24-OHC, 24,25-EC, or CHO at the indicated concentrations for 24 h. Gene expression analysis was conducted as in (A) and (B). Duplicate wells were used for each treatment condition.

Article Snippet: Human hepatoma HepG2 cells and mouse hepatoma AML12 cells were obtained from ATCC.

Techniques: Expressing, Cell Culture, Isolation, Quantitative RT-PCR, Control, Gene Expression