wasp b 9 santa cruz sc 13139  (Santa Cruz Biotechnology)

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: Nature Communications

Article Title: Synthetic aptamer mechanoreceptors enable cell-specific force sensing and temporal control via DNA circuits

doi: 10.1038/s41467-026-70765-w

Figure Lengend Snippet: a Representative brightfield and fluorescence images of HeLa cells treated with internalization inhibitors on AS1411 MP surfaces. b Corresponding quantification of mean fluorescence intensity per cell for HeLa cells. n = 70, 70, 54, 54 (left to right) cells from 3 replicates. Kruskal-Wallis test with Dunn’s multiple comparisons. c Representative images of HeLa cells on AS1411 MP surfaces showing brightfield, mechanosignal, mEGFP-paxillin, and phalloidin-stained actin. d Line profile from ( c ) shows intensity profiles of mechanosignals, mEGFP-paxillin, and actin. e Schematic showing the source of nucleolin-mediated forces. f Quantification of mean fluorescence intensity per cell following cytoskeletal inhibition. n = 72 cells from 3 replicates. Kruskal-Wallis test with Dunn’s multiple comparisons. g Representative brightfield and fluorescent images of HepG2 cells treated with internalization inhibitors on Sgc8 MP surfaces. h Corresponding quantification of mean fluorescence intensity per cell. n = 54 cells from 3 replicates. One-way ANOVA with Bonferroni post-hoc tests. i Representative images showing plasma membrane staining, mechanosignals, and brightfield of HepG2 cells incubated on Sgc8 MP surfaces. j Line profile from ( i ) reveals spatial colocalization of mechanosignals at the inner edge of membrane invaginations. k Schematic showing the source of PTK7-mediated forces. l Brightfield and fluorescence images of HepG2 cells treated with PI3K inhibitor LY294002 on Sgc8 MP surfaces. m Corresponding quantification of mean fluorescence intensity per cell for HepG2 cells. n = 54 cells from 3 replicates. Unpaired, two-tailed Student’s t-test. n Representative brightfield and fluorescence images of HeLa cells after N-WASP siRNA knockdown on AS1411 MP surfaces, and mean fluorescence intensity per cell. n = 85, 83 cells from 3 replicates for scrambled and siRNA groups. Unpaired two-tailed Student’s t-test. o Representative brightfield and fluorescence images of HepG2 cells after N-WASP siRNA knockdown on Sgc8 MP surfaces, and mean fluorescence intensity per cell. n = 45 cells from 3 replicates. Unpaired two-tailed Student’s t-test. All graphs, except ( d , j ), are presented as mean ± s.d. a.u., arbitrary units. Scale bar = 20 µm. Source data are provided as a Source Data file.

Article Snippet: N-WASP antibody (C-1, sc-271484) was purchased from Santa Cruz.

Techniques: Fluorescence, Staining, Inhibition, Clinical Proteomics, Membrane, Incubation, Two Tailed Test, Knockdown