Journal: International Journal of Molecular Sciences

Article Title: COX-2/sEH Dual Inhibitor Alleviates Hepatocyte Senescence in NAFLD Mice by Restoring Autophagy through Sirt1/PI3K/AKT/mTOR

doi: 10.3390/ijms23158267

Figure Lengend Snippet: PTUPB attenuates hepatocyte senescence by enhancing autophagy in AML12 cells. The fluorescence intensity of liver LC3 was detected by immunofluorescence (( A ), scale bars = 50 μm, n = 6). Image J was used to analyze the fluorescence intensity of LC3 (( B ), n = 6). Cells were treated with PTUPB (1 μM) for 1 h before treatment with PA (200 µM). The protein expressions of p62, LC3I/II, and Fundc1 were measured by Western blot and quantitatively analyzed in AML12 cells treated with PA for 48 h (( C – F ), n = 3). Cells were treated with PTUPB (1 μM) and 3-MA (5 mM) for 1 h before treatment with PA (200 µM). The protein expressions of p16, γH2AX, Fundc1, LC3II, and p62 were measured by Western blot and quantitatively analyzed in AML12 cells (( G – L ), n = 3). Data are expressed as the mean ± SD. Differences among multiple groups were performed using ANOVA. Tukey’s test was used as a post hoc test for pairwise comparisons. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Anti-p62 polyclonal antibody , Immunoway , YT7058.

Techniques: Fluorescence, Immunofluorescence, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: COX-2/sEH Dual Inhibitor Alleviates Hepatocyte Senescence in NAFLD Mice by Restoring Autophagy through Sirt1/PI3K/AKT/mTOR

doi: 10.3390/ijms23158267

Figure Lengend Snippet: Antibodies were used in this study.

Article Snippet: Anti-p62 polyclonal antibody , Immunoway , YT7058.

Techniques:

Journal: Molecules

Article Title: Anti-Metabolic Syndrome Effects of Fucoidan from Fucus vesiculosus via Reactive Oxygen Species-Mediated Regulation of JNK, Akt, and AMPK Signaling

doi: 10.3390/molecules24183319

Figure Lengend Snippet: Effects of fucoidan from Fucus vesiculosus (FvF) on relieving insulin resistance (IR) in HepG2 cells. Effects of sodium palmitate (PA) on cellular glucose consumption ( a ). Cells were treated with a concentration range of PA for 24 h. Effects of FvF on glucose consumption in IR cells ( b ). Cells were treated with Metf (2 mM) or FvF (100 μg/mL) in the presence of 100 μM PA for 24 h. ( c ). Reactive oxygen species (ROS) was detected by in situ dihydroethidium (DHE) staining (200×). C, control group; M, cells were treated with 100 μM PA for 24 h; Metf and FvF, cells were treated with metformin (2 mM) or FvF (100 μg/mL) in the presence of 100 μM PA for 24 h. Phosphorylation of c-Jun N-terminal kinase (pJNK) ( d ) and phosphorylation of protein kinase B (pAkt) ( e ) protein levels changed between different treatment groups. C, control group; M, cells treated with 100 μM PA for 24 h; Metf and FvF, cells treated with 100 μM PA for 24 h then incubated with metformin (2 mM) or FvF (100 μg/mL) for another 6 h. Data are expressed as the mean ± SEM. Differences were assessed by ANOVAs and statistical results are denoted as follows: * p < 0.05 versus the control group; # p < 0.05 versus the model group.

Article Snippet: After that, HepG2 cells were rinsed with PBS and lysed in ice-cold RIPA buffer containing protease inhibitor and phosphatase inhibitors (Roche) for 30 min. After denaturation with 5× loading buffer at 100 °C for 10 min, proteins were electrophoresed on 10% SDS-PAGE and transferred to a PVDF membrane (0.22 μm) that was subsequently blocked with 5% ( w / v ) non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 2 h. The membranes were incubated with primary antibodies directed against JNK, pJNK (T183/T185), Akt, pAkt (S473), AMPK, pAMPK (T172), HMGCR, pHMGCR (S872) (Merck Millipore, Billerica, MA, USA), SREBP-1C (Abcam, HongKang, China), ACC, pACC (S79), and β-actin antibodies in 5% ( w / v ) BSA in TBST overnight at 4 °C.

Techniques: Concentration Assay, In Situ, Staining, Control, Phospho-proteomics, Incubation