arp2 3 inhibitor ck 666  (Merck & Co)

Journal: Journal of Extracellular Vesicles

Article Title: Neuronal Extracellular Vesicles Carrying APOE Downregulate Filament Actin Polymerization Signaling to Inhibit Synapse Formation in Alzheimer's Disease

doi: 10.1002/jev2.70248

Figure Lengend Snippet: APOE in APPNEVs impairs synapses by downregulating F‐actin polymerization signaling. (A and B) DIV14 primary neurons are treated for 48 h with DMSO (Vehicle), 10 µM Rac1 inhibitor NSC23766, 5 µM N‐WASP inhibitor Wiskostatin, 30 µM Arp2/3 inhibitor CK666, or 10 µg/mL APPNEVs. Synaptic integrity is assessed by measuring PSD95 protein expression through western blot analysis (A) and immunofluorescence staining (B). (C and D) To further investigate the role of APOE in APPNEVs on actin cytoskeleton regulation, DIV14 primary neurons are treated for 48 h with DMSO (Vehicle), 1 µM EZ‐482, 10 µg/mL APPNEVs, or a combination of 10 µg/mL APPNEVs and 1 µM EZ‐482 (pre‐incubated for 1 h). (C) Rac1 activation is evaluated using a pull‐down assay to isolate Rac1‐GTP, followed by western blot analysis to quantify the levels of active GTP‐bound Rac1 and total Rac1. (D) To assess Arp2/3 complex activation, neurons are immunostained for phosphorylated Arp2 (p‐Arp2). The cytoskeletal structure is visualized using phalloidin staining, while nuclei are stained with DAPI. Phosphorylation levels and cytoskeletal organization are analyzed using confocal microscopy. (E) Neurons are treated for 6 h with 10 µg/mL APPNEVs, with or without 25 nM Rac1 activator ML‐099, and p‐Arp2 levels are detected by western blot. (F) Neurons are incubated with APPNEVs for 48 h, followed by treatment with or without 20 nM Jasplakinolide for the final 20 min. F‐actin in neurons is visualized using phalloidin staining and z‐stack confocal imaging, and the total number of dendritic spines is quantified. Data are presented as mean ± SEM from n = 3–5 independent experiments per condition. Statistical comparisons are performed using one‐way ANOVA followed by Tukey's post‐hoc test, with statistical significance indicates as * p < 0.05 and ** p < 0.01. “ns” indicates no significant change.

Article Snippet: Neurons were cultured for 14 DIV (days in vitro) and treated with 10 μg/mL APPNEVs or WTNEVs, 1 μM of the APOE inhibitor EZ‐482 (HY‐103706, MCE), 10 μM of the Rac1 inhibitor NSC23766 (HY‐15723A, MCE), 5 μM of the N‐WASP inhibitor Wiskostatin (HY‐12534, MCE), 30 μM of the Arp2/3 inhibitor CK666 (HY‐16926, MCE), 25 nM Rac1 activator ML‐099 (HY‐124306; MedChemExpress), or vehicle control.

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Incubation, Activation Assay, Pull Down Assay, Phospho-proteomics, Confocal Microscopy, Imaging

Journal: Journal of Extracellular Vesicles

Article Title: Neuronal Extracellular Vesicles Carrying APOE Downregulate Filament Actin Polymerization Signaling to Inhibit Synapse Formation in Alzheimer's Disease

doi: 10.1002/jev2.70248

Figure Lengend Snippet: Schematic model of APPNEVs carrying APOE downregulate F‐actin polymerization signaling to inhibit synapse formation in AD. During AD progression, EVs derived from APP/PS1 neurons transport APOE into healthy neurons, potentially interacting with neuronal APOE receptors (LRP1, LDLR, VLDLR) to transduction the signaling. This signaling inhibits Rac1‐GTP activation and subsequently downregulates F‐actin polymerization through the Rac1–N‐WASP–Arp2/3 pathway. Disruption of this pathway impairs mature synapse formation, ultimately converting healthy neurons into synaptically damaged neurons and exacerbating AD progression.

Article Snippet: Neurons were cultured for 14 DIV (days in vitro) and treated with 10 μg/mL APPNEVs or WTNEVs, 1 μM of the APOE inhibitor EZ‐482 (HY‐103706, MCE), 10 μM of the Rac1 inhibitor NSC23766 (HY‐15723A, MCE), 5 μM of the N‐WASP inhibitor Wiskostatin (HY‐12534, MCE), 30 μM of the Arp2/3 inhibitor CK666 (HY‐16926, MCE), 25 nM Rac1 activator ML‐099 (HY‐124306; MedChemExpress), or vehicle control.

Techniques: Derivative Assay, Transduction, Activation Assay, Disruption

Journal: Small (Weinheim an Der Bergstrasse, Germany)

Article Title: Lamellipodia‐Mediated Osteoblast Haptotaxis Guided by Fibronectin Ligand Concentrations on a Multiplex Chip

doi: 10.1002/smll.202401717

Figure Lengend Snippet: Effect of small molecules on cell migration and morphology. A) Tracking (top row) and rose plots (bottom row) illustrate changes in directional migration in small molecule groups compared to the haptotaxis control. B) All cell migration analysis in this and subsequent figures is conducted in the Mid FN1 gradient chamber. C) The quantification of percentage of cells migrating upward (toward the fibronectin gradient) or downward (away from the fibronectin gradient). D) Phalloidin‐stained cell areas for the mid fibronectin gradient condition were compared with small molecule groups, including 20 µ m CK666, 100 µ m CK666, and 50 µ m Y27632. N = 80 for all groups. Statistical analyses were performed using a one‐way ANOVA test with Dunnett's multiple comparison test.

Article Snippet: In the multiwell plates, primary osteoblast progenitors were seeded at a density of 30k cells per well and cultured until confluence was reached within 24–48 h. Following this, a straight‐line scratch was made in each well using a 10 μL pipette tip, and the cell monolayer was gently washed with 1X PBS to remove detached cells before replenishing with fresh media or media containing varying concentrations of CK666 (Arp2/3 inhibitor; Cayman Chemical 29038) and of Y27632 (ROCK inhibitor; Selleckchem S1049).

Techniques: Migration, Control, Staining, Comparison

Journal: Small (Weinheim an Der Bergstrasse, Germany)

Article Title: Lamellipodia‐Mediated Osteoblast Haptotaxis Guided by Fibronectin Ligand Concentrations on a Multiplex Chip

doi: 10.1002/smll.202401717

Figure Lengend Snippet: Effect of small molecule inhibitors Y27632 (50 µ m ) and CK666 (20, 100 µ m ) on cell haptotaxis. A) The forward migration index (yFMI), B) velocity, and C) Euclidean distance were compared among groups using estimation plots, along with the one‐way ANOVA with Dunnett's post hoc test. N = 80 for all groups in Figure panel (A–C). D) Analysis of cell protrusion numbers was conducted using a one‐way ANOVA with Dunnett's post hoc test, comparing all groups against the Mid FN1 gradient control. N = 30–32.

Article Snippet: In the multiwell plates, primary osteoblast progenitors were seeded at a density of 30k cells per well and cultured until confluence was reached within 24–48 h. Following this, a straight‐line scratch was made in each well using a 10 μL pipette tip, and the cell monolayer was gently washed with 1X PBS to remove detached cells before replenishing with fresh media or media containing varying concentrations of CK666 (Arp2/3 inhibitor; Cayman Chemical 29038) and of Y27632 (ROCK inhibitor; Selleckchem S1049).

Techniques: Migration, Control

Journal: eLife

Article Title: Damage-induced basal epithelial cell migration modulates the spatial organization of redox signaling and sensory neuron regeneration

doi: 10.7554/eLife.94995

Figure Lengend Snippet: ( A ) Confocal max-projected images of control or CK666-treated transiently injected Tg(Krtt1c19e:Lifeact-mRuby ) larvae. Arrows point to lamellipodia in the control larva, and lack of lamellipodia in the CK666-treated larva. Scale bar = 10 µm. ( B ) Plot of keratinocyte speed over 1 hr post-wound (hpw) as treated in A. N = 10 larvae each collected from three replicates. ( C ) Plot of keratinocyte distance moved over 1 hpw as treated in A. N = 10 larvae each collected from three replicates. ( D ) Confocal sum-projected time-series images of hydrogen peroxide level (pentafluorobenzenesulfonyl fluorescein [Pfbsf] intensity) in 1 larva over 1 hpw in the indicated treatment. ( E ) Quantification of Pfbsf intensity in the wound or fin area of the represented larva after burn injury as treated in D over 1 hpw. N = 1 representative larva per condition. ( F ) Confocal max-projected images of sensory axons 24 hpw in larvae wounded in control medium or CK666. ( G ) Quantification of axon density 24 hpw in larvae treated as in J. N>22 larvae per condition from four replicates. ( H ) Quantification of sensory perception 24 hpw in larvae treated as in J. N = 32 larvae per condition from four replicates. Unless otherwise specified, scale bars = 20 µm. ns = not significant. Figure 4—source data 1. Numerical data for . Figure 4—source data 2. Numerical data for . Figure 4—source data 3. Numerical data for . Figure 4—source data 4. Numerical data for . Figure 4—source data 5. Numerical data for .

Article Snippet: Chemical compound, drug , CK666 (Arp2/3 inhibitor) , Sigma-Aldrich , CAS 442633-00-3 , .

Techniques: Control, Injection

Journal: eLife

Article Title: Damage-induced basal epithelial cell migration modulates the spatial organization of redox signaling and sensory neuron regeneration

doi: 10.7554/eLife.94995

Figure Lengend Snippet:

Article Snippet: Chemical compound, drug , CK666 (Arp2/3 inhibitor) , Sigma-Aldrich , CAS 442633-00-3 , .

Techniques: Software