Journal: bioRxiv

Article Title: β-Amyloid impairs Proteasome structure and function. Proteasome activation mitigates amyloid induced toxicity and cognitive deficits

doi: 10.1101/2024.10.23.619877

Figure Lengend Snippet: The impairment can be mitigated by proteasome activators. (A) 20S Proteasome chymotrypsin-like peptidase activity is inhibited by oligomeric Aβ42, but not by Aβ42 monomers or fibrils. N = 4. Asterisks denote statistically significant differences (p<0.05). Right: atomic force microscopy (AFM) images of Aβ particles (tapping mode in air). The occasional larger particles in the “monomer” preparation are likely spontaneously forming oligomers. ( B ) Morphometric analysis of the 20S proteasome particles imaged by AFM (tapping mode in liquid) reveals shifts in the particles’ dimensions upon incubation with oligomeric Aβ42. 827 control 20S particles (incubated with a vehicle) and 1181 particles incubated with 2 µM oligomeric Aβ42 were analysed. Solid lines are fittings for the frequencies of control (black) and oligo-treated (red) particles. Since almost all particles are in to-view position, the “length” parameter generated during the particle analysis corresponds to the diameter of the 20S α face. The diameters are raw numbers without correction for tip broadening. When the correction of 2 pixels for SNL probe is applied, the diameter for peak 1 (raw: 14 - 15 nm) falls into 10 – 11 nm range, in excellent agreement with the crystal structure of the human 20S proteasome . See Results for putative assignment of proteasome forms to the numbered peaks. (C) Incubation with oligomeric Aβ42 shifts the conformational equilibrium of 20S core particles imaged by AFM (tapping mode in liquid) toward less open-gate and closed-gate forms, but more intermediate forms. (D) Oligomeric Aβ42 does not significantly affect degradation of oxidized hemoglobin. Degradation of hemoglobin is enhanced by a range of oligomeric Aβ42 concentrations. N=4 samples. ( E, F ) Treatment of the 20S proteasome with activators TAT1-DEN or TAT1-TOD partially protects from inhibition inflicted by the oligomeric Aβ42. ( G ) Incubation with the proteasome activator TAT1-DEN induces a dramatic shift toward open-gate forms, even in the presence of 2 µM of oligomeric Aβ42. The numbers in columns indicate percent of conformers. The number of particles analyzed: 733 (vehicle control), 843 (with oligo Aβ42), 270 (with 1 µM TAT1-DEN) and 171 (with oligo Aβ42and TAT1-DEN). Average ± SD, n= 5 to 9 fields.

Article Snippet: Purified 20S Proteasome (R&D Systems, Cat# E-360), purified 26S Proteasome (R&D Systems, Cat# E-365).

Techniques: Activity Assay, Microscopy, Incubation, Control, Generated, Particle Size Analysis, Inhibition

Journal: bioRxiv

Article Title: β-Amyloid impairs Proteasome structure and function. Proteasome activation mitigates amyloid induced toxicity and cognitive deficits

doi: 10.1101/2024.10.23.619877

Figure Lengend Snippet: (A) Native page immunoblot depicting purified 20S and 26S proteasome under incubation with oligomeric Aβ42. Immunoblot performed against proteasome β5 subunit and accompanying total protein silver stain. Arrows depict 26S and 20S proteasome assemblages. (B) Native page immunoblot depicting purified 26S proteasome under incubation with varying concentrations of oligomeric Aβ42. Top image shows a representative set, histogram represents N=3 per condition. (C) Model for impact of Aβ on proteasome processes. *p < 0 . 05, Student’s t test was used unless otherwise stated. N represents the number of animals or samples per group .

Article Snippet: Purified 20S Proteasome (R&D Systems, Cat# E-360), purified 26S Proteasome (R&D Systems, Cat# E-365).

Techniques: Clear Native PAGE, Western Blot, Purification, Incubation, Silver Staining

Journal: Cell death and differentiation

Article Title: A keratinocyte-adipocyte signaling loop is reprogrammed by loss of BTG3 to augment skin carcinogenesis.

doi: 10.1038/s41418-024-01304-7

Figure Lengend Snippet: Fig. 6 Model of BTG3 in the regulation of adipogenesis and the development of skin cancer. Our data are consistent with a role of BTG3 in safeguarding the functional interplay between keratinocytes and adipocytes. In the absence of BTG3, keratinocytes, by releasing IL1α, IL10 and CCL4, promote their own mesenchymal transition by an autocrine mechanism and adipocyte differentiation through paracrine. The latter, in turn, fuels further keratinocyte proliferation and migration by releasing EGF, CCL20, and FGF7, thus forming a feedforward loop to promote skin oncogenesis.

Article Snippet: The following recombinant proteins were used: IL1α (200-LA-002), IL10 (217-IL-005), CCL4 (271-BME-010), CCL20 (360-MP-025), FGF7 (251-KG-010), and IL15 (247-ILB-005) from R&D Systems, and EGF (E4269) from SigmaAldrich.

Techniques: Functional Assay, Migration

Journal: Cellular and Molecular Life Sciences

Article Title: Crosstalk between purinergic receptor P2Y 11 and chemokine receptor CXCR7 is regulated by CXCR4 in human macrophages

doi: 10.1007/s00018-024-05158-7

Figure Lengend Snippet: P2Y 11 and IL-1R cooperate to upregulate CXCR7 surface expression and CCL20 production in primary human M2 macrophages: potentiation by PDE4 inhibition. a M2 macrophages were treated for 24 h with ATPγS (20 µM) alone or in combination with IL-1ß (2 ng/ml) in the presence or absence of rolipram (10 µM). Flow cytometry was used to determine CXCR7 expression (light blue; isotype controls in red). Numbers are mean fluorescence intensities (MFIs) after subtraction of isotype control MFIs. b P2Y 11 receptor antagonist NF340 (20 µM) served to confirm that ATPγS-mediated changes were specific to P2Y 11 receptor activation. c Quantification of CXCR7 expression ( n = 3): mean values ± SD are shown. ** p < 0.01, *** p < 0.001, **** p < 0.0001. D) Quantification of CCL20 production after the same treatments that were used to upregulate CXCR7 expression ( n = 5). Mean values ± SD are shown. **** p < 0.0001

Article Snippet: Levels of CCL20 (MIP-3α) in cell culture supernatants were assessed by ELISA using the Human CCL20/MIP-3 alpha DUO Set ELISA (R&D Systems) according to the manufacturer’s instructions.

Techniques: Expressing, Inhibition, Flow Cytometry, Fluorescence, Control, Activation Assay

Journal: Cellular and Molecular Life Sciences

Article Title: Crosstalk between purinergic receptor P2Y 11 and chemokine receptor CXCR7 is regulated by CXCR4 in human macrophages

doi: 10.1007/s00018-024-05158-7

Figure Lengend Snippet: P2Y 11 /IL-1R induced and rolipram-enhanced CXCR7 upregulation in primary human M2 macrophages is mediated by EGFR. a M2 macrophages were treated for 24 h with P2Y 11 receptor agonist ATPγS plus IL-1ß to induce CXCR7 upregulation. (B) Potentiation of CXCR7 expression by PDE4 inhibitor rolipram. a,b To examine EGFR involvement, EGFR-TKIs AG1478 and erlotinib were used to modulate P2Y 11 /IL-1R induced and rolipram-enhanced CXCR7 upregulation. Representative FACS histograms of CXCR7 expression (light blue; isotype controls in red) are shown in the left panel. Numbers represent mean fluorescence intensities (MFIs) of the respective staining after subtraction of isotype control MFIs. a,b Quantification of CXCR7 expression ( n = 3) and CCL20 production ( n = 5) is shown in the right panel. Data shown are mean values ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Levels of CCL20 (MIP-3α) in cell culture supernatants were assessed by ELISA using the Human CCL20/MIP-3 alpha DUO Set ELISA (R&D Systems) according to the manufacturer’s instructions.

Techniques: Expressing, Fluorescence, Staining, Control

Journal: Cellular and Molecular Life Sciences

Article Title: Crosstalk between purinergic receptor P2Y 11 and chemokine receptor CXCR7 is regulated by CXCR4 in human macrophages

doi: 10.1007/s00018-024-05158-7

Figure Lengend Snippet: CXCR4 antagonists do not downregulate but rather enhance CXCR7 expression and CCL20 production in primary human M2 macrophages. a CXCR7 expression: M2 macrophages were treated for 24 h with P2Y 11 receptor agonist ATPγS plus IL-1ß in the absence (upper panel) or presence of PDE4 inhibitor rolipram (lower panel). CXCR4 antagonists mavorixafor, plerixafor and TC14012 were used to modulate P2Y 11 /IL-1R induced and rolipram-enhanced CXCR7 expression. Flow cytometry was used to determine CXCR7 expression ( n = 3; mean fluorescence intensities, MFIs, after subtraction of isotype control MFIs). Mean values ± SD are shown. * p < 0.05. b CXCR4 antagonists mavorixafor, plerixafor and TC14012 were used to modulate P2Y 11 /IL-1R induced (upper panels; n = 3) and rolipram-enhanced CCL20 production (lower panels; n = 3): data shown are mean values ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Levels of CCL20 (MIP-3α) in cell culture supernatants were assessed by ELISA using the Human CCL20/MIP-3 alpha DUO Set ELISA (R&D Systems) according to the manufacturer’s instructions.

Techniques: Expressing, Flow Cytometry, Fluorescence, Control

Journal: Cellular and Molecular Life Sciences

Article Title: Crosstalk between purinergic receptor P2Y 11 and chemokine receptor CXCR7 is regulated by CXCR4 in human macrophages

doi: 10.1007/s00018-024-05158-7

Figure Lengend Snippet: The P2Y 11 -recombinant astrocytoma cell line naturally expresses CXCR7 but lacks CXCR4: enhancement of CCL20 production through CXCR7 activation and no need for PDE4 inhibition. a Flow cytometry was used to determine P2Y 11 receptor as well as CXCR7 and CXCR4 expression (light blue; the respective isotype controls in red). Numbers are mean fluorescence intensities (MFIs) after subtraction of isotype control MFIs. b Astrocytoma cells and M2 macrophages were treated for 24 h with P2Y 11 receptor agonist ATPγS plus IL-1ß in the absence of PDE4 inhibitor rolipram and CCL20 levels were determined ( n = 5). Mean values ± SD are shown. **** p < 0.0001. c Astrocytoma cells and M2 macrophages were treated for 24 h with P2Y 11 receptor agonist ATPγS plus IL-1ß in the presence of PDE4 inhibitor rolipram and CCL20 levels were determined ( n = 3). Mean values ± SD are shown. * p < 0.05, **** p < 0.0001. d CXCR4 antagonists mavorixafor, plerixafor and TC14012 were used to modulate P2Y 11 /IL-1R induced CCL20 production ( n = 3). Data shown are mean values ± SD. ** p < 0.01, **** p < 0.0001

Article Snippet: Levels of CCL20 (MIP-3α) in cell culture supernatants were assessed by ELISA using the Human CCL20/MIP-3 alpha DUO Set ELISA (R&D Systems) according to the manufacturer’s instructions.

Techniques: Recombinant, Activation Assay, Inhibition, Flow Cytometry, Expressing, Fluorescence, Control

Journal: Cellular and Molecular Life Sciences

Article Title: Crosstalk between purinergic receptor P2Y 11 and chemokine receptor CXCR7 is regulated by CXCR4 in human macrophages

doi: 10.1007/s00018-024-05158-7

Figure Lengend Snippet: CXCR7 knockdown by RNA interference in astrocytoma cells attenuates CCL20 expression. a Astrocytoma cells were transfected with either control siRNA or CXCR7 siRNA for 48 h, with or without subsequent P2Y 11 /IL-1R stimulation for 24 h. The level of knockdown was controlled by measuring CXCR7 surface expression by flow cytometry. Numbers are mean fluorescence intensities (MFIs) after subtraction of isotype control MFIs. b Quantification of CXCR7 knockdown ( n = 4): mean values ± SD are shown. *** p < 0.001. ATPγS/IL-1ß induced and TC14012-enhanced CCL20 production was determined in control cultures (control siRNA) or in cultures subjected to CXCR7 knockdown (CXCR7 siRNA; n = 5): mean values ± SD are shown. **** p < 0.0001. c Cell viability was determined by eFluor 780-based dead cell exclusion ( n = 4): mean values ± SD shown. d Astrocytoma cells were transfected with either control siRNA or IL1R1 siRNA for 48 h, with or without subsequent P2Y 11 /IL-1R stimulation for 24 h. The level of knockdown was controlled by measuring IL-1R1 expression by flow cytometry. Numbers are mean fluorescence intensities (MFIs) after subtraction of isotype control MFIs. e Quantification of IL1-R1 knockdown ( n = 4): mean values ± SD are shown. **** p < 0.0001. ATPγS/IL-1ß induced and TC14012-enhanced CCL20 production was determined in control cultures (control siRNA) or in cultures subjected to IL1R1 knockdown (IL1R1 siRNA; n = 3): mean values ± SD are shown. **** p < 0.0001. f Cell viability was determined by eFluor 780-based dead cell exclusion ( n = 4): mean values ± SD shown

Article Snippet: Levels of CCL20 (MIP-3α) in cell culture supernatants were assessed by ELISA using the Human CCL20/MIP-3 alpha DUO Set ELISA (R&D Systems) according to the manufacturer’s instructions.

Techniques: Knockdown, Expressing, Transfection, Control, Flow Cytometry, Fluorescence

Journal: Cellular and Molecular Life Sciences

Article Title: Crosstalk between purinergic receptor P2Y 11 and chemokine receptor CXCR7 is regulated by CXCR4 in human macrophages

doi: 10.1007/s00018-024-05158-7

Figure Lengend Snippet: Synergistic cooperation between P2Y 11 and CXCR7 in primary human macrophages eliminates the need for IL-1ß co-stimulation and PDE4 inhibition. a M2 macrophages were treated for 24 h with P2Y 11 receptor agonist ATPγS and CXCR7 agonist TC14012, alone or in combination, and CCL20 was determined in culture supernatants ( n = 5). Mean values ± SD are shown. **** p < 0.0001. b Flow cytometry was used to determine CXCR7 and CXCR4 expression (light blue; isotype controls in red). Numbers represent mean fluorescence intensities (MFIs) of the respective staining after subtraction of isotype control MFIs. c Quantification of CXCR7 ( n = 5) and CXCR4 expression ( n = 3). Mean values ± SD are shown. *** p < 0.001, **** p < 0.0001. d M2 macrophages were treated for 24 h with P2Y 11 receptor agonist ATPγS plus IL-1ß in the presence or absence of either CXCR4 antagonist plerixafor or CXCR7 agonist TC14012. Pooled supernatants were analyzed for the presence of 20 CCL chemokines using RayBio technology. Plerixafor- or TC14012-mediated modulation of CCL chemokines that were present in the secretome and produced in a P2Y 11 receptor dependent manner (i.e. sensitive to inhibition with NF340) is shown (see also Fig. )

Article Snippet: Levels of CCL20 (MIP-3α) in cell culture supernatants were assessed by ELISA using the Human CCL20/MIP-3 alpha DUO Set ELISA (R&D Systems) according to the manufacturer’s instructions.

Techniques: Inhibition, Flow Cytometry, Expressing, Fluorescence, Staining, Control, Produced

Journal: Cellular and Molecular Life Sciences

Article Title: Crosstalk between purinergic receptor P2Y 11 and chemokine receptor CXCR7 is regulated by CXCR4 in human macrophages

doi: 10.1007/s00018-024-05158-7

Figure Lengend Snippet: Working model of P2Y 11 crosstalk with IL-1R and chemokine receptors summarizing current findings and knowledge. P2Y 11 couples to G q and G s proteins. While G q activates phospholipase Cß and thus initiates the mobilization of Ca 2+ (via inositol triphosphate, IP 3 ) as well as the activation of protein kinase C (PKC, via diacylglycerol, DAG), G s activates adenylyl cyclase (AC) to increase the levels of cyclic adenosine monophosphate (cyclic AMP). P2Y 11 upregulates CXCR4 and via IL-1R also CXCR7. Upregulation of IL-1R, CXCR7 and CCL20 depends on cyclic AMP. Epidermal growth factor receptor (EGFR) supports upregulation of CXCR7, which serves as an EGFR activator potentiating its signaling capacity, generating a feed-forward loop. Thus, CXCR7 stimulates its own expression with the help of EGFR. By coupling to G i , which blocks the cyclic AMP-generating enzyme AC, and by recruiting the cyclic AMP-degrading enzyme PDE4 via ß-arrestin, CXCR4 serves as a perfectly suited sentinel of intracellular cyclic AMP levels and thus as a regulatory checkpoint of P2Y 11 /IL-1R induced and EGFR/CXCR7-mediated responses (dashed inhibitory arrow) such as the selective activation of CCL20 secretion. Once CXCR7 is fully activated by TC14012, it takes control and eliminates CXCR4 expression (inhibitory arrow) and thus also the need for rolipram-mediated PDE4 inhibition

Article Snippet: Levels of CCL20 (MIP-3α) in cell culture supernatants were assessed by ELISA using the Human CCL20/MIP-3 alpha DUO Set ELISA (R&D Systems) according to the manufacturer’s instructions.

Techniques: Activation Assay, Expressing, Control, Inhibition

Journal: Nature Communications

Article Title: Thermally active nanoparticle clusters enslaved by engineered domain wall traps

doi: 10.1038/s41467-021-25931-7

Figure Lengend Snippet: a Schematic of the experimental setup used to visualize and control the Ferrite Garnet Film (FGF). b Detailed sketch of the FGF with magnetic bubble domains filled by different numbers of paramagnetic nanoparticles. The external magnetic field \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{{{{{{{\bf{B}}}}}}}}}_{{{{{{{{\rm{ext}}}}}}}}}={B}_{z}\hat{{{{{{{{\bf{z}}}}}}}}}$$\end{document} B ext = B z z ^ is applied perpendicular to the film ( z axis). c Polarization microscope image of trapped nanoparticles (of diameter d = 360 nm). The magnetic bubble domains are visible due to the polar Faraday effect. Scale bar is 10 μ m, see also VideoS in the Supporting Information. d Square of the bubble diameter D 2 versus applied field B z . Scattered points are experimental data while continuous line is a linear fit according to \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${D}^{2}=4{a}^{2}[({B}_{z}/{B}_{{{{{{{{\rm{s}}}}}}}}}+1)\sin (\pi /3)/(2\pi )]$$\end{document} D 2 = 4 a 2 [ ( B z / B s + ) sin ( π / 3 ) / ( 2 π ) ] (see “Methods”), from which we extract the lattice constant a = 11.81 ± 0.02 μ m and the saturation magnetization B s = 21.3 ± 0.3 mT. Error bars in D 2 are obtained from the statistical average of different measurments. Insets show images of the magnetic domains. e Three-dimensional view of the magnetostatic potential U calculated at an elevation z = 1.3 μ m and for B z = 0 mT. The ( x , z ) positions are rescaled by a , while the potential U is rescaled by the parameter \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${U}_{0}=\chi \pi {d}^{3}{B}_{{{{{{{{\rm{s}}}}}}}}}^{2}/(12{\mu }_{0})$$\end{document} U 0 = χ π d 3 B s 2 / ( 12 μ 0 ) , see text for the values of μ 0 , χ , and d .

Article Snippet: Above the magnetic lattice we deposit a water dispersion of paramagnetic nanoparticles with diameter d = 360 nm (Microparticles GmbH), and doped with ~40% by weight of iron oxide.

Techniques: Microscopy