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: International Journal of Molecular Sciences

Article Title: Enhanced Therapeutic Potential of the Secretome Released from Adipose-Derived Stem Cells by PGC-1α-Driven Upregulation of Mitochondrial Proliferation

doi: 10.3390/ijms20225589

Figure Lengend Snippet: In vitro validation of the effects of the peroxisome proliferator activated receptor λ coactivator 1α (PGC-secretome) on cell viability and expression of various markers. ( A ) Effects of each secretome on the viability of AML12 hepatocytes. Cell viability analysis revealed that the PGC-secretome increased the viability of thioacetamide (TAA)-treated AML12 hepatocytes significantly more than did the control and normal secretome. ( B ) Western blot analysis demonstrating the effects of each secretome on the expression of various markers in AML12 hepatocytes. The markers included those for liver proliferation (p-STAT3, t-STAT3, VEGF, and HGF), mitochondrial fusion (OPA-1), mitochondrial fission (DRP-1), pro-apoptosis (BIM), and anti-apoptosis (Bcl-xL). PGC-secretome significantly increased the expression of markers related to proliferation, mitochondrial fusion, and anti-apoptosis, and significantly decreased the expression of the markers related to mitochondrial fission and pro-apoptosis. Values are presented as mean ± standard deviation of three independent experiments; * p < 0.05. Abbreviations: Bcl-xL, B-cell leukemia-extra large; BIM, Bcl-2-like protein 11; Ct, control; DRP-1, dynamin related protein 1; HGF, hepatocyte growth factor; NS, normal secretome; OPA-1, Opa1 mitochondrial dynamin like GTPase; PCNA, proliferating cell nuclear antigen; PS, PGC-secretome; TAA, thioacetamide; VEGF, vascular endothelial growth factor.

Article Snippet: A non-tumorigenic AML12 mouse hepatocyte cell line (CRL-2254), was purchased from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: In Vitro, Biomarker Discovery, Expressing, Control, Western Blot, Standard Deviation

Journal: Cell & Bioscience

Article Title: Aldo-keto reductase family 1 member A1 (AKR1A1) exerts a protective function in alcohol-associated liver disease by reducing 4-HNE accumulation and p53 activation

doi: 10.1186/s13578-024-01200-0

Figure Lengend Snippet: AKR1A1 knockdown in AML12 hepatocytes exacerbates lipid accumulation, inflammation, and fibrosis. AKR1A1 knockdown was conducted in AML12 hepatocytes by infecting cells with either a control (sh-control) or an AKR1A1-specific (sh-AKR1A1) shRNA lentiviral clone for 48 h. The shRNA-treated AML12 hepatocytes were then subjected to stimulation with a palmitic acid and oleic acid mixture (P/O) to induce steatosis in the absence or presence of 200 mM ethanol for 72 h. Afterward, western blotting and qRT‒PCR were performed to characterize AKR1A1 expression at both the protein ( A ) and mRNA ( B ) levels. ORO staining was performed to examine lipid accumulation in AML12 hepatocytes ( D ). The extent of lipid accumulation was quantified by extracting the bound ORO dye and detecting the absorbance at 450 nm ( C ). Furthermore, the related marker genes involved in FA transport and synthesis ( Cd36 , Fasn , Acaca , Cyp4a , Cyp2e1 , Lipin1 , Ppar-γ , and Srebp1 ) ( E ) and FA oxidation ( Cpt1α , Acox1 , and Pparα ) ( F ) were examined at the mRNA level. In addition, shRNA-treated AML12 hepatocytes were stimulated with 1 µg/ml of LPS to induce inflammation for 24 h and stimulated with 5 ng/ml TGF-β1 to induce fibrosis for 48 h. After the treatments, qRT‒PCT was performed to characterize the mRNA levels of inflammation-related ( Il-1b , Tnf-α ) ( G ) and fibrosis-related ( Fn1 , α-Sma , Col1a1 , and Timp-1 ) ( H ) marker genes. All values were normalized to the β-actin gene and expressed in relation to the sh-control group. Data are presented as the means ± SD ( n ≥ 3). Statistical differences were marked as follows: # p < 0.05, ## p < 0.01, ### p < 0.001 (compared with the sh-control Ctrl group); * p < 0.05, ** p < 0.01, *** p < 0.001 (compared between the other groups)

Article Snippet: The AML12 cell line, which are hepatocytes isolated from the normal liver of a mouse, was purchased from the Bioresource Collection and Research Center, Hsinchu, Taiwan.

Techniques: Knockdown, Control, shRNA, Western Blot, Expressing, Staining, Marker