Journal: Acta Neuropathologica Communications

Article Title: MRC1 and LYVE1 expressing macrophages in vascular beds of GNAQ p.R183Q driven capillary malformations in Sturge Weber syndrome

doi: 10.1186/s40478-024-01757-4

Figure Lengend Snippet: EC-R183Q promote significant THP1 cell adhesion under static and laminar flow-induced condition. a Fluorescence-labeled THP1 cells were incubated with EC-WT and EC-R183Q under static conditions (N = 10). Adherent cells were quantified after 1 h. P -value was calculated by two-tailed t -test. Phase-contrast images of EC-WT (top) or EC-R183Q (bottom) incubated with THP1 cells (green) at 1 h incubation. Scale bar = 50 µm. b Schematic of live-cell imaging set up. A flow rate of 0.5 ml/min was setup using a tabletop syringe pump with a 20 ml syringe Luer-Lock tip. After 5 min of recording, a switch system was used to deliver pre-stained THP1 cells under continuous uninterrupted flow for 30 min. c Time-lapse imaging of THP1 cells (yellow) adhesion to EC-WT (top), and EC-R183Q (bottom) under laminar flow. Images are at time point = 0, 10, 20, and 30 min. Scale bar = 200 µm. N = 6 independent experiments were performed. d Quantification of THP1 cell adhesion under flow over 10, 20, and 30 min. Mann Whitney test was performed to calculate p-value at each time point. e Proteome profiler cytokine array on conditioned media from EC-WT (top) and EC-R183Q (bottom) after incubation in 2% fetal bovine serum EBM2 media for 24 h. Altered protein levels between EC-WT and EC-R183Q are boxed. Protein levels were quantified by measuring dot intensity using FIJI (right). Three independent experiments were performed. f Intercellular adhesion molecule 1 (ICAM1, grey), UEAI (red), and nuclei counterstaining for DAPI (blue) in the SWS brain sections (n = 4). Scale bar = 50 µm. g Time-lapse imaging of THP1 cell (yellow) adhesion to EC-R183Q treated with IgG2A isotype control (top), and EC-R183Q treated with anti-ICAM1 antibody (bottom) under laminar flow. Images are at time point = 0, 10, 20, and 30 min. Scale bar = 200 µm. N = 5 independent experiments were performed. h Quantification of single cell tracking of THP1 cells under flow over 10, 20, and 30 min. Mann Whitney test was performed to calculate p-value at each time point

Article Snippet: The cytokine array was performed on conditioned media using Proteome Profiler human cytokine array (Cat# ARY005B, R&D Systems) in accordance with the manufacturer protocol.

Techniques: Fluorescence, Labeling, Incubation, Two Tailed Test, Live Cell Imaging, Staining, Imaging, MANN-WHITNEY, Control, Single Cell Tracking

Journal: Journal of Cell Science

Article Title: Myosin-Va restrains the trafficking of Na + /K + -ATPase-containing vesicles in alveolar epithelial cells

doi: 10.1242/jcs.046953

Figure Lengend Snippet: cAMP increases Na+/K+-ATPase activity, protein abundance at the plasma membrane and the distance traveled by the Na+/K+-ATPase-containing vesicles in A549-GFPα1 cells. (A) A549-GFPα1 cells were incubated in the absence (CT) or presence of 50 μM forskolin (FSK) for 10 minutes and the Na+/K+-ATPase activity was measured as 86Rb+ uptake. Graph represents mean ± s.e.m. of three experiments. (B) A549-GFPα1 cells were incubated as in A, and the Na+/K+-ATPase abundance at the basolateral plasma membrane was determined by western blot of the BLM fraction using a specific antibody against GFP. E-cadherin was used as a loading control. Graph represents mean ± s.e.m. of three experiments. A representative western blot is shown. (C) The movement of the GFP-labeled particles was recorded as Metamorph stacks and vesicle trajectories were obtained by single-particle tracking using Metamorph software. Vesicles were randomly selected from those that showed plus-end-directed displacement. Left panel shows a representative image of A549-GFPα1 cells. Arrowhead indicates the vesicle whose trajectory is shown in the right panel before (CT) and after FSK treatment (FSK). (D) Average contour length traveled by the vesicles as a function of time. The black line represents control vesicles; at 60 seconds, upon addition of FSK (red line), the vesicles move at a faster rate. The average contour length is determined by averaging over many trajectories as described in the Materials and Methods. **P<0.01; ***P<0.001. Scale bars: 10 μm and 2 μm (magnified images).

Article Snippet: Cell culture Human A549 (ATCC CCL 185) and HeLa cells (ATCC CCL 2) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 mM HEPES.

Techniques: Activity Assay, Incubation, Western Blot, Labeling, Single-particle Tracking, Software

Journal: Journal of Cell Science

Article Title: Myosin-Va restrains the trafficking of Na + /K + -ATPase-containing vesicles in alveolar epithelial cells

doi: 10.1242/jcs.046953

Figure Lengend Snippet: The three isoforms of myosin-V are expressed in A549 cells. (A) RT-PCR using mRNA obtained from A549 and HeLa cells. Primers used for the amplification are described in supplementary material Table S6. (B) Cell lysates from A549 and HeLa cells were obtained and analyzed by western blot with specific antibodies against the three myosin-V isoforms. A representative western blot is shown. (C) The particulate fraction (100,000 g pellet) of A549-GFPα1 cells was loaded onto a flotation sucrose gradient and eight fractions were recovered. The distribution of the proteins of interest was analyzed by western blotting with specific antibodies. A representative western blot is shown. Rab5 and Rab7 are used as markers of early and late endosomes, respectively. (D) Gradients obtained in C were scanned and the marker content was digitally quantified as indicated. Results are expressed as percentage of the total amount of protein.

Article Snippet: Cell culture Human A549 (ATCC CCL 185) and HeLa cells (ATCC CCL 2) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 mM HEPES.

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Western Blot, Marker

Journal: Journal of Cell Science

Article Title: Myosin-Va restrains the trafficking of Na + /K + -ATPase-containing vesicles in alveolar epithelial cells

doi: 10.1242/jcs.046953

Figure Lengend Snippet: Myosin-Va and myosin-Vc colocalize with Na+/K+-ATPase. (A) A549-GFPα1 cells were incubated in the absence or presence of 50 μM FSK for 10 minutes, basolateral membranes (BLM) and intracellular compartments (IC) were isolated and the Na+/K+-ATPase abundance was determined by western blot using a specific antibody against GFP. E-cadherin and actin were used as loading controls for the BLM and IC fractions, respectively. Graph represents mean ± s.e.m. of three experiments. A representative western blot is shown. (B) The IC fraction of A549-GFPα1 cells was loaded onto a flotation sucrose gradient and eight fractions were recovered. The distribution of the proteins of interest was analyzed by western blotting with specific antibodies. A representative western blot is shown. C+, positive control.

Article Snippet: Cell culture Human A549 (ATCC CCL 185) and HeLa cells (ATCC CCL 2) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 mM HEPES.

Techniques: Incubation, Isolation, Western Blot, Positive Control

Journal: Journal of Cell Science

Article Title: Myosin-Va restrains the trafficking of Na + /K + -ATPase-containing vesicles in alveolar epithelial cells

doi: 10.1242/jcs.046953

Figure Lengend Snippet: The average speed of Na+/K+-ATPase-containing vesicles moving towards the cell periphery is increased in cells expressing a myosin-Va stalk-tail. (A) Live imaging of A549-GFPα1 cells (green) transiently transfected with a dominant-negative myosin-Va that has a m-cherry-tag (red) (m-cherry-DN-Va). The movement of the GFP-labeled particles was recorded. Upper panels show a representative image of the transfected A549-GFPα1 cells. Lower panels show the tracking of the movement of one vesicle before (CT) and after forskolin treatment (FSK). (B) Average contour length traveled by the vesicles in A as a function of time. The black line represents the control vesicles; FSK was added at time 60 seconds and is represented as a red line. (C) Live imaging of A549-GFPα1 cells (green) transiently transfected with a dominant-negative myosin-Vc that has a m-cherry-tag (red) (m-cherry-DN-Vc). The movement of the GFP-labeled particles was recorded. Upper panels show a representative image of the transfected A549-GFPα1 cells. Lower panels show the tracking of the movement of one vesicle before (CT) and after forskolin treatment (FSK). (D) Average contour length traveled by the vesicles in C as a function of time. The black line represents control vesicles; FSK was added at 60 seconds and is represented as the red line. Scale bars: 10 μm and 2 μm (magnified images).

Article Snippet: Cell culture Human A549 (ATCC CCL 185) and HeLa cells (ATCC CCL 2) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 mM HEPES.

Techniques: Expressing, Imaging, Transfection, Dominant Negative Mutation, Labeling

Journal: Journal of Cell Science

Article Title: Myosin-Va restrains the trafficking of Na + /K + -ATPase-containing vesicles in alveolar epithelial cells

doi: 10.1242/jcs.046953

Figure Lengend Snippet: The average speed of Na+/K+-ATPase-containing vesicles moving towards the cell periphery is increased in cells expressing a shRNA against myosin-Va. (A) Live imaging of A549-GFPα1 cells (green) transiently transfected with a shRNA against myosin-Va that has a m-cherry-tag (red) (m-cherry-sh-Va). The movement of the GFP-labeled particles was recorded. Upper panels show a representative image of the transfected A549-GFPα1 cells. Lower panels show the tracking of the movement of two vesicles (arrowheads) under control (CT) conditions. (B) Live imaging of A549-GFPα1 cells (green) transiently transfected with a shRNA against myosin-Vc that has a m-cherry-tag (red) (m-cherry-shRNA-Vc). The movement of the GFP-labeled particles was recorded. Upper panels show a representative image of the transfected A549-GFPα1 cells. Lower panels show the tracking of the movement of two vesicles (arrowheads) under control (CT) conditions (C). Graph represents the average contour length traveled by the vesicles as a function of time, calculated as described in methods. The black line represents the m-cherry-sh-Va vesicles and the red line, the m-cherry-sh-Vc vesicles. (D) A549-GFPα1 cells were transfected with a shRNA against myosin-Va or myosin-Vc, cell lysates were isolated and the myosin-Va (left panel) or myosin-Vc (right panel) abundance was determined by western blot using specific antibodies. E-cadherin and tubulin were used as loading controls. Scale bars: 10 μm and 4 μm (magnified images).

Article Snippet: Cell culture Human A549 (ATCC CCL 185) and HeLa cells (ATCC CCL 2) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 mM HEPES.

Techniques: Expressing, shRNA, Imaging, Transfection, Labeling, Isolation, Western Blot

Journal: Journal of Cell Science

Article Title: Myosin-Va restrains the trafficking of Na + /K + -ATPase-containing vesicles in alveolar epithelial cells

doi: 10.1242/jcs.046953

Figure Lengend Snippet: Dominant-negative myosin-Va mimics cAMP-mediated Na+/K+-ATPase increased activity and recruitment to the plasma membrane in A549-GFPα1 cells. (A) Stable clones expressing myosin-Va tail (DN-Va) and myosin-Vc tail (DN-Vc) were generated as described. Expression of the constructs in the permanent clones was analyzed by western blotting using and antibody against the V5 tag. A representative western blot is shown. (B) A549-GFPα1 cells (CT) and A549-GFPα1 cells permanently transfected with DN-Va and DN-Vc were incubated in the absence or presence of 50 μM FSK for 10 minutes and the Na+/K+-ATPase activity was measured as 86Rb+ uptake. Graph represents mean ± s.e.m. of three different experiments. (C) Control (CT), DN-Va and DN-Vc cells were incubated in the absence or presence of 50 μM FSK for 10 minutes and western blots of the basolateral membrane fraction were performed using a specific antibody against GFP. E-cadherin was used as loading control. A representative western blot is shown. *P<0.05; **P<0.01; n.s., not significant; u.s., unstimulated.

Article Snippet: Cell culture Human A549 (ATCC CCL 185) and HeLa cells (ATCC CCL 2) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 mM HEPES.

Techniques: Dominant Negative Mutation, Activity Assay, Clone Assay, Expressing, Generated, Construct, Western Blot, Transfection, Incubation

Journal: Journal of Cell Science

Article Title: Myosin-Va restrains the trafficking of Na + /K + -ATPase-containing vesicles in alveolar epithelial cells

doi: 10.1242/jcs.046953

Figure Lengend Snippet: Myosin-Va and the Na+/K+-ATPase-containing vesicles colocalize. A549-GFPα1 cells were fixed, permeabilized and blocked. Myosin-Va was visualized by using an anti-myosin-Va antibody and a secondary antibody labeled with Alexa Fluor 568. GFP was directly visualized. Cellular distribution of Na+/K+-ATPase-GFPα1 and myosin-Va was analyzed using a Zeiss LSM 510 laser-scanning confocal microscope and colocalization (blue) was determined using the LSM 510 Meta software.

Article Snippet: Cell culture Human A549 (ATCC CCL 185) and HeLa cells (ATCC CCL 2) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 mM HEPES.

Techniques: Labeling, Microscopy, Software

Journal: Journal of Cell Science

Article Title: Myosin-Va restrains the trafficking of Na + /K + -ATPase-containing vesicles in alveolar epithelial cells

doi: 10.1242/jcs.046953

Figure Lengend Snippet: Microtubules and actin filaments are involved in Na+/K+-ATPase traffic. (A) Live imaging of A549 cells incubated with 10 μM nocodazole for 3 hours. The movement of the GFP-labeled particles was recorded as Metamorph stacks and vesicle trajectories were obtained by single-particle tracking using Metamorph software. Upper panels show a representative immunofluorescence of the microtubule cytoskeleton in control (left) and nocodazole (right) conditions. Lower panel shows the tracking of the movement of one vesicle in control (left) and nocodazole (right) conditions. (B) Live imaging of A549 cells incubated with 5 μM cytochalasin D (Cyto D) for 1 hour. The movement of the GFP-labeled particles was recorded as Metamorph stacks and vesicle trajectories were obtained by single-particle tracking using Metamorph software. Upper panels show a representative immunofluorescence of the actin cytoskeleton under control (left) and cytochalasin D (right) conditions. Lower panel shows the tracking of the movement of one vesicle in control (left) and cytochalasin D (right) conditions. (C) Average contour length traveled by the vesicles as a function of time. The blue line represents the control vesicles; the black line, cells treated with cytochalasin D and the red line, cells treated with nocodazole. Scale bars: 10 μm and 2 μm (inset images).

Article Snippet: Cell culture Human A549 (ATCC CCL 185) and HeLa cells (ATCC CCL 2) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 mM HEPES.

Techniques: Imaging, Incubation, Labeling, Single-particle Tracking, Software, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: Muscle-Saturated Bioactive Lipids Are Increased with Aging and Influenced by High-Intensity Interval Training

doi: 10.3390/ijms20051240

Figure Lengend Snippet: Protein expression in muscle of young and older subjects measured before and after 6 weeks high-intensity interval training. Expression of proteins involved in ( a ) glucose transport and metabolism, ( b ) insulin signalling and ( c ) ceramide and diacylglycerol (DAG) metabolism and lipid transport. Age and training effects: * p < 0.05, † p < 0.01, ‡ p < 0.001. Borderline significance: (*) p < 0.1. Young: n = 13, older: n = 21. Data are means ± SEM. AKT ser473 : AKT phosphorylated at ser 473 , ATGL: Adipose triglyceride lipase, CD36: Cluster of differentiation 36, FABPpm: Fatty acid binding protein plasma membrane, FATP4: Fatty acid transport protein, GS: Glycogen synthase, GP: Glycogen phosphorylase, HKII: Hexokinase II, PKCθ: Protein kinase Cθ, PKCθ ser676 : PKCθ phosphorylated at ser 676 , PP2A: Protein phosphatase 2A, SMS2: Sphingomyelin synthase 2, SphK1: Sphingosine kinase 1, SPT: Serine palmitoyl transferase, SNAP23: Synaptosome associated protein 23.

Article Snippet: Primary antibodies: anti-GLUT4 1:12000 (PA1-1065, Fischer Scientific, Roskilde, Denmark), anti-glycogen synthase 1:4000 (#3893, Cell Signaling, Danvers, MA, USA), anti-glycogen phosphorylase (GP) 1:12000 (As09 455, Agrisera, Vännäs, Sweden), anti-hexokinase II (HKII) 1:1000 (ab104836, Abcam, Cambridge, UK) and anti-synaptosome associated protein (SNAP23) 1:3000 (ab3340, Abcam), anti-serine palmitoyl transferase (SPT) 1:4000 in 5% milk in TBS (ab23696, Abcam), anti-sphingomyelin synthase 2 (SMS2) 1:2000 in 5% milk in TBS (ab103060, Abcam), anti-sphingosine kinase 1 (SphK1) 1:1000 in 5% milk in TBS (ab37980, Abcam), anti-protein phosphatase 2A (PP2A) 1:1000 in 5% milk in TBS (ab32141, Abcam), anti-protein kinase C θ (PKCθ) 1:500 in 5% BSA in TBS (ab110728, Abcam), anti-protein kinase C θ p-ser676 (PKCθ ser676 ) 1:500 in 5% BSA in TBS (ab131479, Abcam), anti-fatty acid transport protein 4 (FATP4) 1:500 in 2.5% BSA in PBS (ab200353, Abcam), anti-fatty acid binding protein (FABPpm) 1:1000 in 5% milk in TBS (ab93928 [3E9], Abcam), anti-AKTpan (AKT) 1:1000 in 5% milk in TBS (#4691, Cell Signaling), anti-AKT p-ser473 (AKT ser473 ) 1:500 in 5% BSA in TBS (#4060, Cell Signaling), anti-CD36 1:3000 in 2.5% milk in TBS (AF1955, R&D Systems, Minneapolis, MN, USA), anti-adipose triglyceride lipase (ATGL) 1:500 in 2.5% milk in TBS (10006409, Cayman Chemical, Ann Arbor, MI, USA).

Techniques: Expressing, Binding Assay, Clinical Proteomics, Membrane