Journal: Nature

Article Title: Restoring metabolism of myeloid cells reverses cognitive decline in ageing

doi: 10.1038/s41586-020-03160-0

Figure Lengend Snippet: Data are mean ± s.e.m. unless otherwise specified. a, Representative immunoblots and quantification of the EP2–AKT–GSK3β–GYS1 signalling pathway in mouse peritoneal macrophages isolated from 6-month-old wild type C57B6/J mice treated with EP2 antagonist C52 (100 nM), EP2 agonist butaprost (100 nM) or PGE2 (100 nM) for 20 h. b, Quantification of a; P < 0.0001 by one-way ANOVA; Tukey’s post hoc test **P = 0.0072, ***P < 0.001, ****P < 0.0001 (n = 6 mice per group). c, Quantification of glycogen levels in human MDMs (age (mean ± s.e.m.) 43.9 ± 3.451 years) treated with EP2 agonist butaprost (100 nM) or EP2 antagonist C52 (100 nM) for 20 h. P < 0.001 by one-way ANOVA; Tukey’s post hoc test ****P < 0.0001 (n = 9 per group). d, LC–MS analysis of human MDMs (age (mean ± s.e.m.) 43.9 ± 3.451 years) treated with C52 (100 nM, 20 h) demonstrates upregulation of proximal glycolytic pathway (G6P, F6P, F-1,6-BP) and GSH and downregulation of UDPG and GSSG. Red circles represent metabolites with fold change >1.5, blue circles with fold change <1.5; q-value < 0.05 with false discovery rate (FDR) correction (n = 6 donors per group). e, Enrichment pathway analysis of d using MetaboAnalyst. f, Schematic depicting U-13C-glucose metabolism via glucose-1P (G1P) and UDP-glucose to glycogen synthesis (yellow shaded box) versus flux towards the pentose phosphate pathway (green shaded box), glycolysis or lactate, and the TCA cycle (blue shaded box) with associated mass-labelled molecules. Glucose is labelled at all 6 carbons (in green; M+6) and is converted via the glycolytic pathway to two molecules of pyruvate (or M+3). Pyruvate is transported into the mitochondria and undergoes oxidative decarboxylation to yield acetyl-CoA (M+2) which enters the TCA cycle. Successive additions of labelled acetyl-CoA through the TCA cycle yield M+2, M+4 and M+6. g, Isotope tracing of U-13C-glucose metabolism was performed in human MDMs (age (mean ± s.e.m.) 42.13 ± 3.674 years) treated with EP2 agonist butaprost (100 nM, 20 h) or EP2 inhibitor C52 (100 nM, 20 h; n = 6 donors per group). Activation of EP2 signalling with butaprost increases incorporation of heavy glucose in glycogen precursors G1P and UDP-glucose and reduces labelling of glycolytic intermediates (F-1,6-BP and pyruvate) as well as TCA cycle intermediates (citrate and succinate); inhibition of EP2 with C52 conversely reduces synthesis of glycogen precursors and increases glycolytic and TCA cycle intermediates. h, Schematic depicting changes in glucose metabolism in Fig. 2h, ​,i.i. i, Representative flow cytometry histograms and corresponding MFI quantification of human MDMs with or without the EP2 inhibitor C52 (100 nM, 20 h) from three independent experiments. Surface levels of anti-inflammatory markers CD206 and CD163 increase with inhibition of EP2 signalling whereas levels of pro-inflammatory markers CD86 and CD64 decrease. **P < 0.01, ***P = 0.002 by two-tailed Student’s t-test (n = 30,000 – 40,000 cells per point; n = 3 donors per group). j, Quantification of phagocytosis of fluorescent E. coli particles in human MDMs treated with EP2 inhibitor C52 (100 nM, 20 h) from two independent experiments. ***P = 0.0002 by two-tailed Student’s t-test (n = 9 donors per group).

Article Snippet: Antibodies and their concentrations are listed as follows (antibody, company, catalogue number, dilution): anti-synapsin (Millipore Sigma; AB1543P; 1:1,000); anti-SNAP25 and anti-SNAP25 (Abcam ab41455 and ab2723, respectively, 1 μg/ml); anti-CamKIIa (pan; Cell Signaling Technology; 3362S; 1:1,000); anti-EP1 and anti-EP3 (Cayman Chemical, 101740 and 101760, respectively, 1:200); anti-EP2 (Abcam, {"type":"entrez-nucleotide","attrs":{"text":"Ab167171","term_id":"45476474","term_text":"AB167171"}} Ab167171 , 1:1,000); anti-EP4 (Santa Cruz, Sc-55596, 1:100); anti-p(Ser473)AKT, anti-AKT (pan), anti-p(Ser9) GSK3β (Cell Signaling Technology, 4060S, 2920S and 9336, respectively, 1:1,000, 1:1,000 and 1:500, respectively); anti-GSK3β (Abcam, ab93926, 1:500); anti-p(Ser641,645,649)GYS1 (Millipore Sigma, 07-817, 1:500); anti-GYS1 (human) and anti-GYS1 (mouse) (Thermo Fisher Scientific, MA5-15802 and MA5-15022, respectively, 1:500 and 1:1,000, respectively); anti-TOM20 and anti-TIM17 (Santa Cruz, sc-17764 and sc-271152, respectively, 1:500); anti-VDAC, anti-PGES and anti-β-tubulin (rb host) (Abcam, ab14734, ab62050 and ab6406, respectively, 1:500); anti-OPA1 (BD Biosciences, 612607, 1:1,000); anti-MFN2 (Abnova, 11325-6A8, 1:1,000); anti-p(Ser616)DRP1 (Cell Signaling Technology, 4494S, 1:250); anti-FIS1 (Proteintech, 10956-1-AP, 1:1,000); anti-COX2 (Cayman Chemical, 160126, 1:1,000); anti-β-tubulin (ms host) (EMD Millipore, 05-661, 1:5,000); and anti-β-actin (Millipore Sigma, A5441, 1:10,000).

Techniques: Western Blot, Isolation, Liquid Chromatography with Mass Spectroscopy, Activation Assay, Inhibition, Flow Cytometry, Two Tailed Test

Journal: Nature

Article Title: Restoring metabolism of myeloid cells reverses cognitive decline in ageing

doi: 10.1038/s41586-020-03160-0

Figure Lengend Snippet: Data are mean ± s.e.m. unless otherwise specified. a, MFI quantification from three independent experiments for the anti-inflammatory markers CD71 and EGR2 and pro-inflammatory markers CD80 and CD86 in young (3–4 months) and aged (20–23 months) Cd11bCre and Cd11bCre;EP2lox/lox mice (n = 10,000–20,000 cells per point; n = 3 mice per group). Two-way ANOVA: effects of genotype and age P < 0.0001; Tukey’s post hoc test **P < 0.01, ***P < 0.001, ****P < 0.0001. b, c, Quantification of immune factors in plasma (b) and hippocampus (c) comparing aged Cd11bCre and aged Cd11bCre;EP2lox/lox mice; *P < 0.05, **P < 0.01 by two-tailed Student’s t-test (n = 6 mice per group). d, Representative immunoblot and quantification from two independent experiments comparing EP2 levels in 6-month-old Cd11bCre and wild-type mice (n = 6 per group). e, Primary latency in the Barnes maze for the five learning trials (n = 10 (young), n = 11 (aged Cd11bCre), n = 7 (aged Cd11bCre;EP2lox/lox). f, Representative immunoblot and quantification of hippocampal presynaptic proteins synapsin and SNAP-25 and postsynaptic proteins PSD95 and CamKIIα; #P = 0.0005, ***P = 0.0006, ****P < 0.0001 by two-tailed Student’s t-test (n = 6 mice per group; 20–23-month-old mice). g, Input/output curves as a measure of basal synaptic transmission in the CA1 region of the hippocampus (n = 8 slices, 3 mice per group). h, PGE2 activation of the EP2 receptor activates AKT signalling through phosphorylation of Ser473. Activated pAKT (Ser473) inactivates GSK3β through inhibitory phosphorylation of Ser9. Inactivation of GSK3β leads to constitutive activity of glycogen synthase 1 (GYS1) and glycogen synthesis. Conversely, deletion or antagonism of EP2 receptor signalling leads to downstream inhibitory phosphorylation of Ser641, Ser645 and Ser649 on GYS1 by activated, non-phosphorylated GSK3β. i, Quantification of EP2, pAKT (Ser 473)/total AKT, pGSK3β (Ser9)/total GSK3β and pGYS1 (Ser 641, 645, 649)/GYS1 levels in peritoneal macrophages from 6-month-old Cd11bCre and Cd11bCre;EP2lox/lox mice; ***P = 0.0002, ****P < 0.0001 by two-tailed Student’s t-test (n = 6 mice per group).

Article Snippet: Antibodies and their concentrations are listed as follows (antibody, company, catalogue number, dilution): anti-synapsin (Millipore Sigma; AB1543P; 1:1,000); anti-SNAP25 and anti-SNAP25 (Abcam ab41455 and ab2723, respectively, 1 μg/ml); anti-CamKIIa (pan; Cell Signaling Technology; 3362S; 1:1,000); anti-EP1 and anti-EP3 (Cayman Chemical, 101740 and 101760, respectively, 1:200); anti-EP2 (Abcam, {"type":"entrez-nucleotide","attrs":{"text":"Ab167171","term_id":"45476474","term_text":"AB167171"}} Ab167171 , 1:1,000); anti-EP4 (Santa Cruz, Sc-55596, 1:100); anti-p(Ser473)AKT, anti-AKT (pan), anti-p(Ser9) GSK3β (Cell Signaling Technology, 4060S, 2920S and 9336, respectively, 1:1,000, 1:1,000 and 1:500, respectively); anti-GSK3β (Abcam, ab93926, 1:500); anti-p(Ser641,645,649)GYS1 (Millipore Sigma, 07-817, 1:500); anti-GYS1 (human) and anti-GYS1 (mouse) (Thermo Fisher Scientific, MA5-15802 and MA5-15022, respectively, 1:500 and 1:1,000, respectively); anti-TOM20 and anti-TIM17 (Santa Cruz, sc-17764 and sc-271152, respectively, 1:500); anti-VDAC, anti-PGES and anti-β-tubulin (rb host) (Abcam, ab14734, ab62050 and ab6406, respectively, 1:500); anti-OPA1 (BD Biosciences, 612607, 1:1,000); anti-MFN2 (Abnova, 11325-6A8, 1:1,000); anti-p(Ser616)DRP1 (Cell Signaling Technology, 4494S, 1:250); anti-FIS1 (Proteintech, 10956-1-AP, 1:1,000); anti-COX2 (Cayman Chemical, 160126, 1:1,000); anti-β-tubulin (ms host) (EMD Millipore, 05-661, 1:5,000); and anti-β-actin (Millipore Sigma, A5441, 1:10,000).

Techniques: Two Tailed Test, Western Blot, Transmission Assay, Activation Assay, Activity Assay

Journal: Nature

Article Title: Restoring metabolism of myeloid cells reverses cognitive decline in ageing

doi: 10.1038/s41586-020-03160-0

Figure Lengend Snippet: Experiments were performed in young (3–4-month-old) and aged (20–23-month-old) Cd11bCre and Cd11bCre;EP2lox/lox mice. Data are mean ± s.e.m. unless otherwise specified. a, Schematic depicting the object location memory task. b, Fraction of time spent exploring the displaced object is expressed as the per cent preference for the displaced object. Paired t-test, #P = 0.0280, *P = 0.0188, **P = 0.0034 (n = 9 young, n = 11 aged Cd11bCre, n = 7 aged Cd11bCre;EP2lox/lox mice). c, Representative traces of paths to target hole (green) by aged mice on the day of testing in the Barnes maze. d, Quantification of percentage of time in the target quadrant and distance travelled to the target hole in the Barnes maze. Two-tailed unpaired Student’s t-test, ***P = 0.0003, (n = 10 young, n = 11 aged Cd11bCre, n = 7 aged Cd11bCre;EP2lox/lox mice). e, Long-term potentiation, measured as the change in field excitatory postsynaptic potential (fEPSP), in the CA1 hippocampal region over 120 min. Three episodes of theta-burst stimulation (3 × TBS; black arrows) were applied. Two-way ANOVA, effects of time and genotype P < 0.0001; Sidak’s multiple comparisons test with Geisser–Greenhouse correction, *P = 0.0272 (n = 8 slices, n = 3 mice per group). f, Representative immunoblots of 2 independent experiments showing EP2 signalling via the AKT–GSK3β–GYS1 pathway in peritoneal macrophages from 6-month-old Cd11bCre and Cd11bCre;EP2lox/lox mice (n = 6 mice per group). g, Glycogen levels in peritoneal macrophages from aged Cd11bCre and Cd11bCre;EP2lox/lox mice. Two-tailed Student’s t-test, **P = 0.0031 (n = 6 mice per group). h, Mice were administered 13C-glucose (1 g per kg by oral gavage) and peritoneal macrophages were isolated 4 h later for isotope tracing. i, Quantification of 13C-glucose metabolism in mouse peritoneal macrophages demonstrates a shift away from glycogen synthesis and towards glycolysis, the pentose phosphate pathway (PPP) and the TCA cycle in aged Cd11bCre;EP2lox/lox (cKO) mice relative to Cd11bCre (+/+) mice (n = 6 mice per group). G1P, glucose 1-phosphate; G6P, glucose 6-phosphate; F-1,6BP, fructose 1,6-bisphosphate; pyruv., pyruvate; 6PG, 6-phosphogluconate; R5P, ribulose 5-phosphate.

Article Snippet: Antibodies and their concentrations are listed as follows (antibody, company, catalogue number, dilution): anti-synapsin (Millipore Sigma; AB1543P; 1:1,000); anti-SNAP25 and anti-SNAP25 (Abcam ab41455 and ab2723, respectively, 1 μg/ml); anti-CamKIIa (pan; Cell Signaling Technology; 3362S; 1:1,000); anti-EP1 and anti-EP3 (Cayman Chemical, 101740 and 101760, respectively, 1:200); anti-EP2 (Abcam, {"type":"entrez-nucleotide","attrs":{"text":"Ab167171","term_id":"45476474","term_text":"AB167171"}} Ab167171 , 1:1,000); anti-EP4 (Santa Cruz, Sc-55596, 1:100); anti-p(Ser473)AKT, anti-AKT (pan), anti-p(Ser9) GSK3β (Cell Signaling Technology, 4060S, 2920S and 9336, respectively, 1:1,000, 1:1,000 and 1:500, respectively); anti-GSK3β (Abcam, ab93926, 1:500); anti-p(Ser641,645,649)GYS1 (Millipore Sigma, 07-817, 1:500); anti-GYS1 (human) and anti-GYS1 (mouse) (Thermo Fisher Scientific, MA5-15802 and MA5-15022, respectively, 1:500 and 1:1,000, respectively); anti-TOM20 and anti-TIM17 (Santa Cruz, sc-17764 and sc-271152, respectively, 1:500); anti-VDAC, anti-PGES and anti-β-tubulin (rb host) (Abcam, ab14734, ab62050 and ab6406, respectively, 1:500); anti-OPA1 (BD Biosciences, 612607, 1:1,000); anti-MFN2 (Abnova, 11325-6A8, 1:1,000); anti-p(Ser616)DRP1 (Cell Signaling Technology, 4494S, 1:250); anti-FIS1 (Proteintech, 10956-1-AP, 1:1,000); anti-COX2 (Cayman Chemical, 160126, 1:1,000); anti-β-tubulin (ms host) (EMD Millipore, 05-661, 1:5,000); and anti-β-actin (Millipore Sigma, A5441, 1:10,000).

Techniques: Two Tailed Test, Western Blot, Isolation

Journal: International Journal of Molecular Sciences

Article Title: Airborne Particulate Matter (PM 10 ) Inhibits Apoptosis through PI3K/AKT/FoxO3a Pathway in Lung Epithelial Cells: The Role of a Second Oxidant Stimulus

doi: 10.3390/ijms21020473

Figure Lengend Snippet: Levels of pAKT Ser473 , pAKT Thr308 , and pFoxO3a Ser253 proteins. Representative blot of ( A ) levels of pAKT Ser473 assessed; ( B ) densitometry of pAKT Thr308 protein levels using ImageJ software; ( C ) assessed pAKT Th308 levels; ( D ) densitometry of pAKT Ser473 protein levels using ImageJ software; ( E ) assessed pFoxO3a Ser253 levels; and ( F ) densitometry of pFoxO3a Ser253 protein levels using ImageJ software. Lung epithelial cells were exposed to PM 10 (10 µg/cm2) for 24 h, treated with H 2 O 2 (500 µM) for 24 h, and treated with LY294002 inhibitor (LY) (50 μM) for 1 h before the H 2 O 2 (500 µM) treatment. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein was used as loading control for western blot. The image is representative of three independent experiments, and values are the mean ± SD of three independent experiments.

Article Snippet: Then, 30 μg of protein was loaded in a 12% SDS-PAGE for detection of AKT, as well as phosphorylated pAKT Ser473 and pAKT Thr308 (2920S, 4060S, and 13038S, respectively; Cell Signaling, Danvers, Massachusetts, United States), the transcription factor FoxO3a (ab12162; Abcam, Cambridge, Massachusetts, United States) and phosphorylated pFoxO3a Ser253 (PA5-36816; Thermo Fisher, Rockford, Illinois, United States), and finally catalase and p27 kip1 (12098S, and D69C12, respectively; Cell Signaling, Danvers, Massachusetts, United States).

Techniques: Software, Control, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Airborne Particulate Matter (PM 10 ) Inhibits Apoptosis through PI3K/AKT/FoxO3a Pathway in Lung Epithelial Cells: The Role of a Second Oxidant Stimulus

doi: 10.3390/ijms21020473

Figure Lengend Snippet: Representative blot of ( A ) pAKT Ser473 and total AKT, as well as ( C ) pAKT Thr308 and total AKT assessed by western blot and densitometry of ( B ) pAKT Ser473 and ( D ) pAKT Thr308 of levels using ImageJ software. Lung epithelial cells were pre-exposed to PM 10 (10 µg/cm 2 ) for 24 h, and then cells were treated with H 2 O 2 (500 mM) for 24 h. In lane 3 and 6 of the blot (panel A) are cells treated with LY294002 inhibitor (50 μM) for 1 h before the H 2 O 2 (500 µM) treatment. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein was used as loading control for western blot. pAKT Ser473 ** p < 0.001 versus control; ** p < 0.001 versus PM 10 + H 2 O 2 versus PM 10 + LY + H 2 O 2 . pAKT Thr308 *** p < 0.0001 versus control; *** p < 0.0001 versus PM 10 + H 2 O 2 versus PM 10 + LY + H 2 O 2 . The image is representative of three independent experiments, and values are the mean ± SD of three independent experiments.

Article Snippet: Then, 30 μg of protein was loaded in a 12% SDS-PAGE for detection of AKT, as well as phosphorylated pAKT Ser473 and pAKT Thr308 (2920S, 4060S, and 13038S, respectively; Cell Signaling, Danvers, Massachusetts, United States), the transcription factor FoxO3a (ab12162; Abcam, Cambridge, Massachusetts, United States) and phosphorylated pFoxO3a Ser253 (PA5-36816; Thermo Fisher, Rockford, Illinois, United States), and finally catalase and p27 kip1 (12098S, and D69C12, respectively; Cell Signaling, Danvers, Massachusetts, United States).

Techniques: Western Blot, Software, Control

Journal: BMC biotechnology

Article Title: Optimized zymogram protocol from 3D spheroid cultures to study MMP-2 and -9 activities in tumor cells.

doi: 10.1186/s12896-025-00961-x

Figure Lengend Snippet: Fig. 5 Effect of kidney cancer spheroid number on AKT signaling. p-AKT (Ser 473) analysis in 786-O (A-B) or WT-CLS1 (C-D) spheroids stimulated or not with collagen I. Representative p-AKT immunoblots obtained with total protein extracts from 786-O (upper A) or WT-CLS1 (upper C) spheroids. p-AKT is shown as a 60 kDa protein. Lower A and C, representative Ponceau S staining of the membrane. For each independent experiment, band intensities were quantified using Fiji software (B and D) and normalized to the corresponding Ponceau S staining bands seen on the membrane. Values for a single spheroid were arbitrarily set to 1. Data are presented as the mean +/- SEM of 5 to 6 independent experiments, (n = 6 for 786-O and n = 5 for WT-CLS1 spheroid cultures), *p < 0.05, Kruskal-Wallis test, followed by Dunn’s Multiple Comparison Test

Article Snippet: Then, the membrane was incubated overnight at 4 °C under agitation with a mouse anti-AKT antibody (1:1000; Cell Signaling, ref#2920S) in TBS-T 5% BSA.

Techniques: Western Blot, Staining, Membrane, Software, Comparison