Journal: Iranian Journal of Basic Medical Sciences

Article Title: Curcumin protects human umbilical vein endothelial cells against high oxidized low density lipoprotein-induced lipotoxicity and modulates autophagy

doi: 10.22038/IJBMS.2021.59969.13297

Figure Lengend Snippet: Curcumin ameliorated autophagy inhibition induced by high ox-LDL in HUVECs through the AMPK/mTOR/p70S6K pathway. A and B: The protein expression levels of LC3-II were measured by Western blotting (n=3). C and D: The protein expression levels of AMPK and p-AMPK were measured by Western blotting (n=3). E and F: The protein expression levels of mTOR and p-mTOR were measured by Western blotting (n=3). G and H: The protein expression levels of p70S6K and p-p70S6K were measured by Western blotting. Expression of these proteins was quantified by densitometry using ImageJ software (n=3). The data are shown as the means±SDs (* P < 0.01 vs. the control group; ** P < 0.01 vs. the control group; # P <0.05 vs. the high ox-LDL group; ## P < 0.01 vs. the high ox-LDL group; & P < 0.05 vs. the compound C group). AMPK, AMP-activated protein kinase; mTOR, mammalian target of rapamycin; p70S6K, p70 ribosomal S6 protein kinase

Article Snippet: Protein extracts were isolated, separated on a 12.5% SDS-PAGE gel and transferred onto a 0.22 μm PVDF membrane (Epizyme), which was incubated with the following primary antibodies: peroxisome proliferator-activated receptor γ (PPARγ) (16001-1-AP, Proteintech, Wuhan, China), interleukin-6 (IL-6) (ab233706, Abcam, Cambridge, 1:1000), interleukin-10 (IL-10) (ab133575, Abcam, 1:5000), tumor necrosis factor alpha (TNF-α) (17590-1-AP, Proteintech, Wuhan, China), microtubule-associated protein 1 light chain 3 (LC3) (2775S, Cell Signaling Technology, MA, USA, 1:1000), AMPK (HN0506, HuaBio, Hangzhou, China, 1:1000), mTOR (HN0824, HuaBio, 1:500), p70S6K (HJ0505, HuaBio, 1:1000), phosphorylated AMPK (p-AMPK) (bs-8813R, Bioss, Beijing, China, 1:1000), phosphorylated mTOR (p-mTOR) (2971S, Cell Signaling Technology, 1:500), and phosphorylated p70S6K (p-p70S6K) (9234S, Cell Signaling Technology, 1:1000). β-Actin (AC026, ABclonal, Wuhan, China, 1:50000) was used as an internal control.

Techniques: Inhibition, Expressing, Western Blot, Software

Journal: Viruses

Article Title: The Function behind the Relation between Lipid Metabolism and Vimentin on H9N2 Subtype AIV Replication.

doi: 10.3390/v14081814

Figure Lengend Snippet: Figure 4. The cellular AMPK expression and phosphorylation level. AMPK protein expressions and phosphorylation in HeLa and KO HeLa cells with or without viral infection were detected with Western blotting. (A) The expression and phosphorylation of AMPK protein on KO HeLa cells compared with HeLa cells. (B) The expression and phosphorylation of AMPK protein on HeLa cells after viral infection. (C) The expression and phosphorylation of AMPK protein on KO HeLa cells after viral infection.

Article Snippet: GAPDH monoclonal antibody was purchased from Enogene Biologicals (Nanjing, China). β-actin monoclonal antibody, AMPK monoclonal antibody and AMPK phosphorylated monoclonal antibody P-AMPK were purchased from BOSTER Biologicals (Nanjing, China).

Techniques: Expressing, Phospho-proteomics, Infection, Western Blot

Journal: Viruses

Article Title: The Function behind the Relation between Lipid Metabolism and Vimentin on H9N2 Subtype AIV Replication.

doi: 10.3390/v14081814

Figure Lengend Snippet: Figure 7. Model of lipid metabolism and vimentin on H9N2 subtype AIV. When vimentin was knocked down, the phosphorylation level of AMPK was decreased, resulting in the decreased level of HMGCR phosphorylation, which increased the enzyme activity, thereby increasing cholesterol. After the destruction of lipid rafts, the virus binding was inhibited, and cholesterol helped to stabilize the structure of lipid rafts, which might help the virus bind to cells.

Article Snippet: GAPDH monoclonal antibody was purchased from Enogene Biologicals (Nanjing, China). β-actin monoclonal antibody, AMPK monoclonal antibody and AMPK phosphorylated monoclonal antibody P-AMPK were purchased from BOSTER Biologicals (Nanjing, China).

Techniques: Phospho-proteomics, Activity Assay, Virus, Binding Assay

Journal: Metabolism: clinical and experimental

Article Title: Serum metabolomics analysis reveals increased lipid catabolism in mildly hyperbilirubinemic Gilbert's syndrome individuals.

doi: 10.1016/j.metabol.2021.154913

Figure Lengend Snippet: Fig. 4. AMPK, PPARα, PGC1α and lipid catabolism. The figure presents the % increase of AMPK, PPARα and PGC-1α measured in PBMCs of GS vs. control individuals, as previously published by Mölzer et al. [22] and correlation coefficients (r) between UCB, AMPK; PPARα and PGC-1 α. The table shows correlations coefficients and p-values of AMPK (blue), PGC1α (orange), PPARα (green) and UCB (yellow) with their downstream lipid metabolites in the whole study population using Pearson or Spearman-Rho. Significant correlations are in bold type, t = trend.

Article Snippet: The following antibody set-up was used: rabbit anti-human monoclonal to AMPK α1 (phos-T183) and AMPKα2 (phos-T172) (ab133448, Abcam) and secondary antibody: goat anti-rabbit IgG H & L AlexaFluor 488 (ab150077, Abcam); rabbit anti-human polyclonal to PgC1α PE-labelled (orb124814, Biorbyt) and rabbit anti-human polyclonal to PPARα (phos-Ser12) FITClabelled (bs-4055R-FITC, Bioss).

Techniques: Control

Journal: Molecular Metabolism

Article Title: Exercise-induced specialized proresolving mediators stimulate AMPK phosphorylation to promote mitochondrial respiration in macrophages

doi: 10.1016/j.molmet.2022.101637

Figure Lengend Snippet: SPMs stimulate AMPKα phosphorylation to enhance mitochondrial metabolism in macrophages. ( A ) Representative immunoblot and quantification (right) of phosphorylated AMPKα (Thr172) and total AMPK in BMDMs treated without or with 1 nM RvD1 for 1h. ( B – E ) BMDMs treated without or with 500 nM Compound-C (CompC) and 1 nM of ( B )RvD1, ( C ) RvD2, ( D ) RvE1, or ( E ) MaR1 for 1h. After treatment, cells were subjected to a mitochondrial stress assay and OCR values assessed (top panels B – E ) and derived mitochondrial respiratory parameters were quantified (below). Data expressed as mean ± SEM; n = 3 ( A ) or n = 3–6 ( B – E ); ∗ P < 0.05, ∗∗ P < 0.01; two-tailed Student's t -test (a) or Two-way ANOVA with Holm-Šídák post-test ( B – E ).

Article Snippet: Cells were then stained with a polyclonal, rabbit anti-mouse FITC-labeled p-AMPKα1(Thr172) antibody (Biorbyt, cat. # orb8540) in a 100 μL staining volume (perm/wash buffer) and incubated 30 min on ice.

Techniques: Western Blot, Derivative Assay, Two Tailed Test

Journal: Molecular Metabolism

Article Title: Exercise-induced specialized proresolving mediators stimulate AMPK phosphorylation to promote mitochondrial respiration in macrophages

doi: 10.1016/j.molmet.2022.101637

Figure Lengend Snippet: 5-lipoxygenase derived lipid mediators contribute to exercise-induced mitochondrial function via AMPK activation. ( A ) Flow cytometric analysis (MFI) of intracellular AMPKα1(Thr172) phosphorylation in PMs isolated from WT and Alox5 −/− mice following exercise. ( B ) FlowJo V10.8.1 generated t -SNE map ( B,C ) depicted in a 2D dimensionally reduced dataspace showing all events combined from each experimental groups. The color axis overlay in ( B ) depicts metacluster populations identified using FlowSOM, an unbiased machine learning clustering algorithm. ( C ) The percent frequency of each FlowSOM population (highlighted in red) for all samples combined, is denoted atop of the respective t -SNE map. ( D ) The fluorescence distribution of each surface marker for the respective FlowSOM metacluster population is shown in red (vs the distribution for all cells shown in grey) in the histogram overlays. ( E ) MFI of TMRM for each FlowSOM metacluster population from WT and Alox5 −/− mice following exercise. Data expressed as mean ± SEM; n = 5 ( A and E ); ∗ P < 0.05; two-tailed Student's t -test.

Article Snippet: Cells were then stained with a polyclonal, rabbit anti-mouse FITC-labeled p-AMPKα1(Thr172) antibody (Biorbyt, cat. # orb8540) in a 100 μL staining volume (perm/wash buffer) and incubated 30 min on ice.

Techniques: Derivative Assay, Activation Assay, Isolation, Generated, Fluorescence, Marker, Two Tailed Test