Journal: Nature Communications

Article Title: Early-life exercise extends healthspan but not lifespan in mice

doi: 10.1038/s41467-025-61443-4

Figure Lengend Snippet: a KEGG enrichment analysis identifying the effects of early-life exercise on metabolic pathways in aged mice (18 mo). Size and color of dot indicate number and significance of genes mapped to specific pathways. Significant ( p < 0.05) pathways are shown with color. Rich factor refers to the ratio of DEG numbers annotated in this pathway term to all gene numbers annotated in this pathway term. b Gene set enrichment analysis showing relative gene expression involved in lipid and fatty acid metabolic processes in muscle (left) and liver (right) tissues from aged mice based on 18 mo RNA-seq data. c Pathway diagrams involving glycolysis, fatty acid transport, lipolysis, lipogenesis, fatty acid oxidation, TCA cycle and oxidative phosphorylation, showing gene expression changes induced by early-life exercise in muscles and livers from aged mice (18 mo). d Immunoblotting analyses of lipid metabolic proteins and mitochondrial function biomarkers in muscles and livers from young (4 mo) and old (18 mo) male mice ( n = 6). FASN, fatty acid synthase; CPT1A/B, carnitine palmitoyltransferase 1A/B; ACADL, acyl-Coenzyme A dehydrogenase, long chain; PGC-1α, PPARγ coactivator-1α. e Representative records of O 2 consumption rate and quantified respiration of permeabilized muscle fibers from male mice (16 mo) using octanoylcarntine and malate as substrates. Adenosine diphosphate (ADP) was added to stimulate fatty acid oxidation respiration. To achieve maximal physiological oxidative phosphorylation capacity, succinate was added ( n = 3). f Representative records of O 2 consumption rate and quantified respiration of permeabilized muscle fibers from male mice (16 mo) using pyruvate as substrates ( n = 3). Data are presented as mean ± SEM. Data are analyzed using hypergeometric test ( a ), permutation test ( b ) and two-way ANOVA with Šídák’s multiple comparisons test ( d and e ).

Article Snippet: The membranes were incubated overnight at 4 °C with appropriate primary antibodies followed by incubation with the corresponding secondary antibodies at room temperature for 2 h. The primary antibodies were as follows: FASN (FASN, 3180S, Cell Signaling Technology), PPARγ coactivator-1α (PGC-1α, 2178S, Cell Signaling Technology), Atrogin 1 (67172-1-Ig, Proteintech), complex I (Invitrogen, 438800), complex II (Invitrogen, 459200), complex III (Invitrogen, 457125), complex V (Invitrogen, 459240), CD36 (18836-1-AP, Proteintech), carnitine palmitoyltransferase 1B (CPT1B, 22170-1-AP, Proteintech), carnitine palmitoyltransferase 1 A (CPT1A, 15184-1-AP, Proteintech), acyl-Coenzyme A dehydrogenase, long chain (ACADL, 17526-1-AP, Proteintech), p53 (10442-1-AP, Proteintech), p21 (28248-1-AP, Proteintech), p16 (554079, BD Pharmingen), phospho-p70S6K (Thr389) (9234, Cell Signaling Technology), total p70S6K (2708, Cell Signaling Technology), phospho-Akt (Ser473) (4060, Cell Signaling Technology), Akt (9272, Cell Signaling Technology), GAPDH (2118, Cell Signaling Technology) and α -tubulin (2125S, Cell Signaling Technology). α-Tubulin was used as a loading control.

Techniques: Gene Expression, RNA Sequencing, Phospho-proteomics, Muscles, Western Blot

Journal: Redox Biology

Article Title: Sirt1-Sirt3 axis regulates human blood-brain barrier permeability in response to ischemia

doi: 10.1016/j.redox.2017.09.016

Figure Lengend Snippet: Sirt1 regulates Sirt3 expression through AMPK-PGC1 pathway after OGD. Cells were transfected with siRNA for 48 h or pretreated with salermide (50 μM) for 30 min, and exposed to OGD. The expression of Sirt1 and Sirt3 was detected by western blot (A) and calculated (B and C). Cells were pretreated with salermide (50 μM) or compound C (5 μM) for 30 min and exposed to OGD. The expression of Sirt1 and Sirt3 was detected by western blot (D) and calculated (E and F). Cells were transfected with siRNA for 48 h with or without compound C, or pretreated with salermide (50 μM) for 30 min with or without compound C, and exposed to OGD. The expression of Sirt3 was detected by western blot (G). Cells were transfected with Si-control or Si-PGC1 for 48 h and exposed to OGD. The expression of Sirt3 was detected by western blot (H). Data are shown as mean ± SEM. * p < 0.05 vs. Control. # p < 0.05 vs. OGD. & p < 0.05 vs. Si-control. $ p < 0.05.

Article Snippet: The specific siRNA targeted Sirt1 (Si-Sirt1, sc-40986), Sirt3 (Si-Sirt3, sc-61555), PGC1 (Si-PGC1, sc-38884) and control siRNA (Si-Control, sc-37007), which should not knock down any known proteins, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Transfection, Western Blot, Control

Journal: Frontiers in oncology

Article Title: The Pan-Sirtuin Inhibitor MC2494 Regulates Mitochondrial Function in a Leukemia Cell Line.

doi: 10.3389/fonc.2020.00820

Figure Lengend Snippet: FIGURE 4 | (A) mRNA evaluation of PGC1α in U937 cells treated with MC2494 at 50 µM concentration for 3 and 24 h. U937 cells were treated with MC2494 for the indicated times at 50 µM concentration. GAPDH was used as control for equal loading. Graphs show the mean of at least 2 independent experiments with error bars indicating standard deviation. (B) Top panel, western blot analysis of PGC1α; bottom panel western blot analysis of PGC1β. U937 cells were treated with MC2494 at 50 µM concentration for indicated times. Numbers on Western blot indicate the results of densitometry analysis, performed using the Image J Gel Analysis tool. (C) Immunofluorescence for PGC1α. Bar, 10 µM. Immunofluorescence microscopy analysis (right graph) is representative of 3 independent experiments. Values are mean ± standard deviation (SD) of biological triplicates. ***p ≤0.001, **p ≤0.01 vs. control cells.

Article Snippet: Antibodies: PGC1α (#ab191838), PGC1β (#ab176328), and SOD2 (#ab13533) were from Abcam and GAPDH was from Santa Cruz (#sc-47724).

Techniques: Concentration Assay, Control, Standard Deviation, Western Blot, Microscopy

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Global protease activity profiling provides differential diagnosis of pancreatic cysts

doi: 10.1158/1078-0432.CCR-16-2987

Figure Lengend Snippet: (A) Heatmap displaying cleavage of 30 mucinous-specific substrates following treatment of a mucinous cyst fluid sample with DMSO or various broad-spectrum protease inhibitors. Spectral counts were used for relative quantification of peptide cleavage products. Vertical bar (|) indicates the site of cleavage within substrates. (B) Label-free quantitation of aspartyl protease relative abundance in mucinous (M) and nonmucinous (NM) cysts. (C) Western blot analysis of recombinant (r) and cyst fluid-derived cathepsin E and gastricsin. Samples were pre-incubated at the indicated pH for 10 minutes.

Article Snippet: For recombinant gastricsin (R&D Systems, 6186-AS), cathepsin D (R&D Systems, 1014-AS), and cathepsin E (R&D Systems, 1294-AS), the MSP-MS assay was performed as described above with slight modifications: 10 nM of recombinant protease in pH 3.5 acetate buffer was used and aliquots were removed after 15, 60, and 240 minutes.

Techniques: Quantitation Assay, Western Blot, Recombinant, Derivative Assay, Incubation

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Global protease activity profiling provides differential diagnosis of pancreatic cysts

doi: 10.1158/1078-0432.CCR-16-2987

Figure Lengend Snippet: Histological analysis of mucinous cysts with low-grade dysplasia (A, B, E, F) and high-grade dysplasia (C, D, G, H). Gastricsin (A, C), cathepsin E (E, G), and haematoxylin and eosin (H&E) staining (B, D, F, H) in IPMNs (A–F) and MCNs (G, H). Scale bar is 10 µm.

Article Snippet: For recombinant gastricsin (R&D Systems, 6186-AS), cathepsin D (R&D Systems, 1014-AS), and cathepsin E (R&D Systems, 1294-AS), the MSP-MS assay was performed as described above with slight modifications: 10 nM of recombinant protease in pH 3.5 acetate buffer was used and aliquots were removed after 15, 60, and 240 minutes.

Techniques: Staining

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Global protease activity profiling provides differential diagnosis of pancreatic cysts

doi: 10.1158/1078-0432.CCR-16-2987

Figure Lengend Snippet: (A) Substrate specificity of cathepsin D, cathepsin E, and gastricsin as determined by MSP-MS. Residues shown in iceLogo are statistically significant with p < 0.05. (B) Heatmap comparing the amino acid enrichment Z-scores for gastricsin relative to cathepsin D and cathepsin E. (C) Venn diagram depicting the unique and overlapping cleavages detected by MSP-MS with cathepsin D, cathepsin E, and gastricsin. (D) Cleavage of the fluorescent substrates by cathepsin D, cathepsin E, and gastricsin. Activity was normalized to 1.00 based on the maximal activity against each substrate. Red arrow indicates the site of cleavage. Error bars denote standard error of the mean (SEM) from triplicate analysis.

Article Snippet: For recombinant gastricsin (R&D Systems, 6186-AS), cathepsin D (R&D Systems, 1014-AS), and cathepsin E (R&D Systems, 1294-AS), the MSP-MS assay was performed as described above with slight modifications: 10 nM of recombinant protease in pH 3.5 acetate buffer was used and aliquots were removed after 15, 60, and 240 minutes.

Techniques: Activity Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Global protease activity profiling provides differential diagnosis of pancreatic cysts

doi: 10.1158/1078-0432.CCR-16-2987

Figure Lengend Snippet: Analysis of gastricsin (A) and cathepsin E (B) activity in nonmucinous (NM) and mucinous (M) cysts using fluorescent substrates. (C) ROC curves comparing sensitivity and specificity of CEA, gastricsin, cathepsin E, and CEA and gastricsin in combination.

Article Snippet: For recombinant gastricsin (R&D Systems, 6186-AS), cathepsin D (R&D Systems, 1014-AS), and cathepsin E (R&D Systems, 1294-AS), the MSP-MS assay was performed as described above with slight modifications: 10 nM of recombinant protease in pH 3.5 acetate buffer was used and aliquots were removed after 15, 60, and 240 minutes.

Techniques: Activity Assay