pbluescript ks  (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

Journal: Aging (Albany NY)

Article Title: Lactobacillus amylovorus KU4 inhibits adipocyte senescence in aged mice through necdin regulation of p53 activity

doi: 10.18632/aging.206314

Figure Lengend Snippet: Necdin regulates p53 acetylation and activity by releasing p300 from p53. ( A , B ) Reporter gene analysis using p21-promoter–Luc and the indicated expression plasmids in HEK293T cells treated with 100 μM H 2 O 2 , 50 μM sirtinol, and LKU4–CM for 24 h. ( C ) Immunoprecipitation (IP) analyses of p53 and p300 in HEK293T cells transfected with p53, p300, and NDN expression plasmids with or without LKU4–CM treatment for 24 h. ( D ) p53 acetylation in day 8 3T3-L1 adipocytes transfected with the indicated plasmids and treated with LKU4–CM for 24 h. Cells were immunoprecipitated using an anti-p53 antibody. ( E ) Chromatin immunoprecipitation (ChIP) assay using anti-p53 and anti-p300 antibodies in day 6 3T3-L1 adipocytes transfected with p53, p300, and NDN, and treated with LKU4–CM for 24 h. ( F ) mRNA and protein levels of p21 in 3T3-L1 adipocytes transfected with p300 and NDN expression plasmids and treated with LKU4–CM for 24 h. The lowercase letters above the graphs indicate statistical significance at p < 0.05.

Article Snippet: Plasmids, pBluescript II KS (+)–p21 promoter Luc (p21-promoter–Luc), were purchased from Addgene (Watertown, MA, USA). pCDNA3–p53 and pCDNA3–p300 were transfected into HEK293T cells, 3T3-L1 adipocytes or primary adipocytes. pCDNA3–NDN was constructed for a previous study.

Techniques: Activity Assay, Expressing, Immunoprecipitation, Transfection, Chromatin Immunoprecipitation

Journal: Aging (Albany NY)

Article Title: Lactobacillus amylovorus KU4 inhibits adipocyte senescence in aged mice through necdin regulation of p53 activity

doi: 10.18632/aging.206314

Figure Lengend Snippet: LKU4 negatively regulates H 2 O 2 -induced adipocyte senescence through NDN upregulation. ( A ) Reporter gene analysis using p21-promoter–Luc in HEK293T cells. ( B – G ) γH2AX, p53, p21, and NDN protein expression ( B ), SA-β-gal staining (scale bars, 200 μm) ( C ), RT-qPCR analysis of SASP genes ( D ) and mitochondrial function-associated genes ( E ), ROS levels ( F ), mtDNA copy number and CS activity ( G ) in primary adipocytes. Primary adipocytes differentiated from SVF cells were transfected with expression plasmids and NDN siRNA for 12 h, followed by treatment with 100 μM H 2 O 2 , 50 μM sirtinol, and LKU4–CM for another 24 h, as indicated, before analysis. ( H ) Oxygen consumption rate (OCR) analysis using a Seahorse XFe analyzer in 3T3-L1 adipocytes overexpressing the indicated expression plasmids in the absence or presence of LKU4–CM. ( I ) Intracellular TG levels in primary adipocytes. Differentiated primary adipocytes were transfected with expression plasmids and NDN siRNA and then treated with 100 μM H 2 O 2 , 50 μM sirtinol, and LKU4–CM, as indicated.

Article Snippet: Plasmids, pBluescript II KS (+)–p21 promoter Luc (p21-promoter–Luc), were purchased from Addgene (Watertown, MA, USA). pCDNA3–p53 and pCDNA3–p300 were transfected into HEK293T cells, 3T3-L1 adipocytes or primary adipocytes. pCDNA3–NDN was constructed for a previous study.

Techniques: Expressing, Staining, Quantitative RT-PCR, Activity Assay, Transfection