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