Journal: BMC biology

Article Title: Lipid metabolites affected by deficient autophagy antagonize the occurrence of autophagy through AMPK signaling in insects.

doi: 10.1186/s12915-025-02274-z

Figure Lengend Snippet: Fig. 7 Effects of metabolites on autophagy occurrence in Drosophila fat body and B. mori BmN cells. A and B LysoTracker Red staining (A), western blots and protein quantification of BmAtg8–PE and p-AMPKα (T172) (B) of Drosophila fat body at W stage after treatment with 40 μM LPE(19:0), LPC(0:0/17:0), LPC(0:0/19:0), or LPC(20:0/0:0) for 2 h, individually. C LysoTracker Red staining from nutritious and starved BmN cells after treatment with LPE(19:0), LPC(0:0/17:0), LPC(0:0/19:0), or LPC(20:0/0:0) (40 μM) for 2 h (N, normal nutrient; S, starvation). D-G Western blots and protein quantification of BmAtg8–PE and p-AMPKα (T172) after treatment with 40 μM LPE(19:0) (D), LPC(0:0/17:0) (E), LPC(0:0/19:0) (F), or LPC(20:0/0:0) (G) for 2 h in normal-nutrition and starved BmN cells (S, starvation). The solvent PBS was used as control. Data are presented as means ± SD; P values were determined by unpaired two-tailed Student’s t-test. Significance test was performed between control and each metabolite treatment. *P < 0.05, **P < 0.01, ***P < 0.001. ns, no significant difference

Article Snippet: The primary antibodies for V5 (Cell Signaling Technology, 13202S; 1:3000), FLAG (Cell Signaling Technology, 14793S; 1:3000), HA (Santa Cruz Biotechnology, sc-7392; 1:2000), BmAtg8–PE (Abcam, ab109364; 1:4000), Phospho-AMPKα (Thr172) (Cell Signaling Technology, 50081; 1:3000), and Tubulin (Beyotime Biotechnology, AT819; 1:5000) were used for western blotting according to the standard procedure as previously described [17, 41].

Techniques: Staining, Western Blot, Solvent, Control, 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: 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