Journal: Disease Markers

Article Title: Urolithin B, a Gut Microbiota Metabolite, Reduced Susceptibility to Myocardial Arrhythmic Predisposition after Hypoxia

doi: 10.1155/2022/6517266

Figure Lengend Snippet: Urolithin B activates the PI3K/Akt pathway to inhibit hypoxia-induced cardiomyocyte apoptosis and suppressed nuclear translocation of NF- κ B to facilitate nerve remodeling. (a–c) Western blotting results and densitometric analysis of p-Akt, T-Akt, p-mTOR, and T-mTOR protein levels in the hypoxia group and the hypoxia+urolithin B group. ∗ p < 0.05; n = 5 per group (Student's t -test). (d–f) Western blotting results and densitometric analysis of bax and caspase-3 protein levels in the hypoxia+urolithin B and hypoxia+urolithin B+Akti group. ∗ p < 0.05; n = 5 per group (Student's t -test). (g–i) Western blotting results and densitometric analysis of NF- κ B and IkBa protein levels in the hypoxia group and the hypoxia+urolithin B group. ∗ p < 0.05; n = 5 per group (Student's t -test).

Article Snippet: Membranes were further probed with primary antibodies against phosphorylated (p)-Akt (1 : 1000; CST #4060), total (t)-Akt (1 : 1000; CST #9272), phosphorylated (p)-mTOR (1 : 1000; CST #5536), total (t)-mTOR (1 : 1000; CST #2972), bcl2 (1 : 1000; CST #3498), bax (1 : 1000; CST #5023), caspase-3 (1 : 1000; CST #14220), Cx43 (1 : 1000; CST #3512), GAP-43 (1 : 1000; CST #8945), TH (1 : 1000; CST #2792), IL-6 (1 : 1000; Abcam #ab233706)), NF- κ B (1 : 1000; CST #8242), IkBa (1 : 1000, CST #4812), and β -actin (1 : 1000, CST #4970) at 4°C overnight.

Techniques: Translocation Assay, Western Blot

Journal: Toxicology letters

Article Title: Damage of uremic myocardium by p-cresyl sulfate and the ameliorative effect of Klotho by regulating SIRT6 ubiquitination.

doi: 10.1016/j.toxlet.2022.06.006

Figure Lengend Snippet: Fig. 1. Klotho inhibited the downregulation of SIRT6 protein expression, cardiomyocyte hypertrophy, and phosphorylation of mTOR induced by PCS. (A) NRCMs were exposed to various concentrations of PCS (50, 250, and 500 μmol/L) for 48 h, and then, the mRNA expression of BNP and β-MHC was analyzed by qPCR. (B) NRCMs were pretreated with Klotho (1, 10, and 100 μg/L) for 1 h and then exposed to PCS (500 μmol/L) for 48 h, and the expression of BNP and β-MHC was measured by qPCR. (C) Treatment of NRCMs with 100 μg/L Klotho (pretreated for 1 h) inhibited the decrease in SIRT6 protein expression induced by 500 μmol/L PCS, which was measured by Western blotting. (D) Klotho or PCS intervention did not affect SIRT6 mRNA expression, as analyzed by qPCR. (E) NRCMs were transfected with siSIRT6 or scrambled siRNA (siCntl), pretreated with 100 μg/L Klotho for 1 h, and then incubated with PCS (500 μmol/L) for an additional 48 h, and the mRNA expression of BNP and β-MHC was measured by qPCR. (F) The cell size was determined by immunofluorescence staining using a troponin antibody (400×). (G) NRCMs were transfected with siSIRT6 or scrambled siRNA (siCntl), pretreated with 100 μg/L Klotho for 1 h, and then incubated with PCS (500 μmol/L) for an additional 48 h, and the expression of p-mTOR and t-mTOR was measured by Western blotting.

Article Snippet: A rabbit anti-total mTOR (t-mTOR) antibody, rabbit anti-phosphorylated mTOR (p-mTOR) antibody, rabbit anti-SIRT6 antibody, rabbit anti-ubiquitinspecific peptidase 10 (USP10) antibody, rabbit anti-carboxyl terminus of Hsp70-interacting protein (CHIP) antibody, rabbit anti-E3 ubiquitin ligase (UBE3A) antibody, and mouse anti-ubiquitin antibody were purchased to Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Phospho-proteomics, Western Blot, Transfection, Incubation, Immunofluorescence, Staining

Journal: Toxicology letters

Article Title: Damage of uremic myocardium by p-cresyl sulfate and the ameliorative effect of Klotho by regulating SIRT6 ubiquitination.

doi: 10.1016/j.toxlet.2022.06.006

Figure Lengend Snippet: Fig. 7. Schematic view of the damaging effect of PCS on cardiomyocytes. PCS induces ubiquitination of SIRT6 and downregulates the protein expression of SIRT6, which leads to phosphorylation of mTOR and DNA DSBs in cardiomyocytes. Ultimately, PCS induces cardiomyocyte hypertrophy and apoptosis. Klotho protects cardiomyocytes from the effects of PCS by inhibiting the ubiquitination of SIRT6 and blocking the downregulation of SIRT6 protein expression. PCS, p-cresyl sulfate

Article Snippet: A rabbit anti-total mTOR (t-mTOR) antibody, rabbit anti-phosphorylated mTOR (p-mTOR) antibody, rabbit anti-SIRT6 antibody, rabbit anti-ubiquitinspecific peptidase 10 (USP10) antibody, rabbit anti-carboxyl terminus of Hsp70-interacting protein (CHIP) antibody, rabbit anti-E3 ubiquitin ligase (UBE3A) antibody, and mouse anti-ubiquitin antibody were purchased to Cell Signaling Technology (Danvers, MA, USA).

Techniques: Ubiquitin Proteomics, Expressing, Phospho-proteomics, Blocking Assay

Journal: Nutrition & Metabolism

Article Title: Prenatal inflammation causes obesity and abnormal lipid metabolism via impaired energy expenditure in male offspring

doi: 10.1186/s12986-022-00642-y

Figure Lengend Snippet: A, B The mRNA expression level of DEGs in RNA-seq analysis. C, D The mRNA expression level of glucogenesis, lipogenesis, lipid synthesis and lipolysis in liver and fat tissue mice (n = 5 per genotype). E, F Western blot and quantification of p-mTOR/t-mTOR, PPAR-γ, FAS, G-6-P and PCK in liver. Data are mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 by unpaired Student t test or Mann–Whitney U test. Pla2g1b phospholipase A2g4a, Fabp4 fatty acid binding protein 4, Lpl lipoprotein lipase, Cel carboxyl ester lipase, Ceslg carboxylesterase 1G, Fasn fatty acid synthase, G-6-p glucose 6 phosphatase, Ucp1 uncoupling protein-1, Pnliprp1 pancreatic lipase related protein 1, Pnlip pancreatic lipase, Clps colipase

Article Snippet: The primary antibodies used were β-actin (#3700), phosphorylated (p)-mTOR (Ser2448) (# 5536S), total (t)-mTOR (#2983) and Peroxisome proliferator-activated receptor-γ (PPAR-γ) (#2435 T) (Cell Signaling Technology, MA, US).

Techniques: Expressing, RNA Sequencing Assay, Western Blot, MANN-WHITNEY, Binding Assay