u6 expression vectors  (Addgene inc)

Journal: Biofactors (Oxford, England)

Article Title: Lactobacillus johnsonii JNU3402 Ameliorates Age‐Related Liver Dysfunction Through Stimulating PGC ‐1α‐Mediated SIRT1 Expression

doi: 10.1002/biof.70069

Figure Lengend Snippet: LJ3402 prevents hepatic senescence and mitochondrial dysfunction in aged mice. (A) Representative images of hematoxylin and eosin (H&E) (top), SA‐β‐gal (middle), and Oil Red O (bottom) staining on frozen liver sections from the indicated group. (B) The mRNA levels of marker genes involved in senescence ( p16 , p21 , p53 ) and protein levels (γH2AX, p21, p53) in the liver of young and aged mice were determined by RT‐qPCR or immunoblotting ( n = 3; three randomly selected samples per group). (C) Expression of marker genes involved in lipid metabolism ( Srebp1c , Pparα , Cpt1 ) and mitochondrial homeostasis ( Sirt1 , Pgc‐1α , NRF1 , TFAM ) in the liver was determined using RT‐qPCR. (D) Hepatic triglyceride (TG), total cholesterol (TC), and free fatty acid (FFA), and (E) plasma levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) ( n = 5; three randomly selected samples per group). (F) Livers separated from mice were incubated in culture medium, and the media were collected after 12 h and 24 h of incubation; the concentrations of TNF‐α and IL‐1β in the media were measured ( n = 5; three randomly selected samples per group). All data are presented as the mean ± SEM. * p < 0.05 and ** p < 0.01. Different lowercase letters above the bars indicate statistically significant differences ( p < 0.05).

Article Snippet: The pBluescript II KS(+)‐p21 promoter‐luc plasmid was purchased from Addgene (MA, USA) and designated as p21 promoter‐luc.

Techniques: Staining, Marker, Quantitative RT-PCR, Western Blot, Expressing, Clinical Proteomics, Incubation

Journal: Biofactors (Oxford, England)

Article Title: Lactobacillus johnsonii JNU3402 Ameliorates Age‐Related Liver Dysfunction Through Stimulating PGC ‐1α‐Mediated SIRT1 Expression

doi: 10.1002/biof.70069

Figure Lengend Snippet: SIRT1 modulates LJ3402‐mediated suppression of p53 function in senescent AML12 hepatocytes. (A) Acetylation of p53 after 5 days of treatment with LJ3402‐CM (LJ‐CM) and 50 μM sirtinol during H 2 O 2 ‐induced senescence. (A, C–I) Senescence of AML12 hepatocytes was induced by 1 mM or 750 μM H 2 O 2 for 1 h per day for 7 days. LJ‐CM, 50 μM sirtinol, or 20 μM PFTα were added on days 3–7 during H 2 O 2 exposure. (B) Luciferase reporter activity in HEK293T cells transfected with a reporter plasmid ( p21 promoter–luc) and a p53 expression plasmid. Twelve hours after transfection, cells were treated with LJ‐CM and 50 μM sirtinol for 24 h with or without 100 μM H 2 O 2 . (C) The mRNA and protein levels of p21, p53, PGC‐1α, and SIRT1 measured by RT‐qPCR and immunoblotting, respectively. (D) SA‐β‐gal staining. (E) mRNA levels of genes involved in mitochondrial biogenesis ( NRF1 and TFAM ) and oxidant defense ( CAT and SOD ). (F) ROS levels. (G) Oxygen consumption rates (OCRs) measured at baseline and after treatment with 2.5 μM oligomycin, 1 μM FCCP, or 1 μM rotenone/antimycin A. (H) Oil Red O staining. (I) Intracellular TG levels in senescent AML12 hepatocytes treated with LJ‐CM, sirtinol, and PFTα. All data are presented as the mean ± SEM. Different lowercase letters above the bars indicate statistically significant differences ( p < 0.05).

Article Snippet: The pBluescript II KS(+)‐p21 promoter‐luc plasmid was purchased from Addgene (MA, USA) and designated as p21 promoter‐luc.

Techniques: Luciferase, Activity Assay, Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Western Blot, Staining