ck666  (Tocris)

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: bioRxiv

Article Title: Targeting GL-Lect driven endocytosis to suppress cell plasticity in breast cancer

doi: 10.64898/2026.01.08.698324

Figure Lengend Snippet: A) ECAD internalization was monitored by IF in MCF10A-EPN3 cells treated with AP2µ KD or mock. Top, representative images; internalized ECAD (green), DAPI (blue). Bar, 20 µm. Bottom, quantification of relative internalized ECAD fluorescence intensity/cell in individual field of views, normalized to mock control. N (fields of view): Mock=4, AP2µ=35; n=3. B) ECAD internalization in MCF10A-EPN3 cells subjected to single or double Eps15/Eps15L1 KDs. Representative images and quantification as in (A). Bar, 20 µm. N (fields of view): Mock=42, Eps15 KD=39, Eps15L1 KD=40, Double KD=35, n=3. C) Left panels: PM, representative immuno-EM images showing PM-ECAD-positive (gold-labeled) tubular invaginations (indicated by arrows) in MCF10A-EV and MCF10A-EPN3 cells; scale bar, 200 nm; Int, representative immuno-EM images showing internalized ECAD-positive (gold-labeled) structures (indicated by arrows and enlarged in the insets) in MCF10A-EV and MCF10A-EPN3 cells; scale bar, 250 nm. Right upper panel: quantification of internalized ECAD expressed as a percentage of PM-ECAD. N (cells): EV=24, EPN3=27. Right-lower panel: gold-labelled ECAD-positive clathrin-coated pits (CCPs) and tubular invaginations (TIs) expressed as percentage of total number of structures in 100 µm PM length/cell. N (cells): EV=27, EPN3=25. D) Effects of inhibitors on ECAD internalization in MCF10A-EPN3 cells. Cells were pre-treated with the indicated compounds or vehicle (DMSO) before measuring ECAD internalization as in (A): CK666 (50 µM, 1 h), Genz-123346 (4 µM, 6 days), Lactose (100 mM, 1h), I3 (20 µM, 10 min). Representative images and quantification as in (A). Bar, 20 µm. N (fields of view): CK666=42 (DMSO control=44) (n=5); Genz=20 (DMSO control=20) (n=3); Lactose=33 (mock=40) (n=3); I3=40 (DMSO control=53) (n=6). E) ECAD internalization in MCF10A-EPN3 cells subjected to I3 treatment as in (D), Gal3 KD or Gal3 KD/I3 treatment. Representative images and quantification as in (A). Bar, 20 µm. N (fields of view): Mock=34, I3=23, Gal3 KD=26, Gal3 KD/I3=28, n=2. F) Co-internalization of Gal3–ECAD was monitored for 10 min in MCF10A-EV and -EPN3 cells. Left: representative confocal images, Gal3-Alexa488 (green), anti-ECAD (red), DAPI (blue). Bar: 20 µm. Right: Manders overlap coefficient of internalized Gal3-ECAD. N (cells): EV/EPN3=69; n=2. In all panels, results are shown as mean±SD, except panel for E in which median ± max/min values are shown. p-values (unpaired Student’s t-test, two-tailed): **** <0.0001; *** <0.001; ** <0.01; * <0.05, ns, not significant.

Article Snippet: The following inhibitors of endocytic players were used in this study: CK666 (Arp2/3 complex inhibitor, 50 μM, SML0006 Merck), EIPA (NHE inhibitor, 50 μM; used as macropinocytosis inhibitor, A3085 Merck), I3 (non-permeable Galectin-3 inhibitor, 20 μM, GalectoBiotech), Genz-123346 (ceramide synthase inhibitor, 4 μM, 5.38285 Merck), and (+)-Lactose (pan-galectin inhibitor, 100 mM, L3750 Merck).

Techniques: Fluorescence, Control, Labeling, Two Tailed Test

Journal: bioRxiv

Article Title: Targeting GL-Lect driven endocytosis to suppress cell plasticity in breast cancer

doi: 10.64898/2026.01.08.698324

Figure Lengend Snippet: A) Internalization of Tf-488 was monitored in MCF10A-EV and -EPN3 cells with or without AP2µ KD. Results are shown normalized to mock control. N (fields of view): EV, Mock=56, AP2µ KD=28 (n=3); EPN3, Mock=44, AP2µ KD=28 (n=3). B) Quantification of Tf-488 internalization in MCF10A-EV and - EPN3 cells subjected to single or double Eps15/Eps15L1 KDs. N (fields of view): EV, Mock=35, Eps15 KD=35, Eps15L1 KD=35, Double KD=35 (n=3); EPN3, Mock=36, Eps15 KD=35, Eps15L1 KD=35, Double KD=35 (n=3). C) Negative controls for inhibitor screening (supporting ). Quantification of Tf-488 internalization in MCF10A-EV and -EPN3 cells pre-treated with the following compounds or vehicle control: CK666 (50 µM, 1 h), Genz (4 µM, 6 days), I3 (20 µM, 10 min). N (fields of view): EV, DMSO=15, CK666=15, Genz=15, I3=15 (n=3); EPN3, DMSO=15, CK666=15, Genz=15, I3=14 (n=3). D-E) Positive controls for inhibitor screening (supporting ). (D) CD44 internalization was monitored in vivo by IF using an anti-CD44 antibody in MCF10A-EV and -EPN3 cells pre-treated with I3 (20 µM, 10 min) or vehicle control. Quantification of relative CD44 fluorescence intensity is shown normalized to control. N (fields of view): EV, DMSO=18, I3=16; EPN3, DMSO=18, I3=16 (n=3). (E) Shiga-toxin (STXB) endocytosis and binding was monitored by continuous incubation of STXB-488 conjugated ligand in MCF10A-EV and- EPN3 cells pre-treated with Genz (4 µM, 6 days). Quantification of relative STXB fluorescence intensity is shown normalized to control. N (fields of view): EV, DMSO=12, Genz=12; EPN3, DMSO=12, Genz=12 (n=3). F) Quantification of Transferrin internalization in MCF10A-EV and -EPN3 cells subjected to I3 (20 µM, 10 min) treatment, Gal3 KD or Gal3 KD/I3 treatment. N (fields of view): EV, mock=20, I3=20, Gal3 KD=20, Gal3 KD/I3=20 (n=2); EPN3, mock=21, I3=20, Gal3 KD=21, Gal3 KD/I3=21 (n=2). G) Quantification of CD44 internalization in MCF10A-EV and -EPN3 cells subjected to I3 (20 µM, 10 min) treatment, Gal3 KD or Gal3 KD/I3 treatment. N (fields of view): EV, mock=20, I3=20, Gal3 KD=21, Gal3 KD/I3=20 (n=2); EPN3, mock=20, I3=19, Gal3 KD=20, Gal3 KD/I3=20 (n=2). H) ECAD internalization was monitored in MCF10A-EV cells subjected to Eps15/Eps15L1 single or double KD. Quantification of relative ECAD fluorescence intensity/cell normalized to mock control. N (fields of view): Mock=42, Eps15 KD=39, Eps15L1 KD=37 and Double KD=40 (n=3). I-M) ECAD internalization was monitored in MCF10A-EV cells pre-treated as in panel C. Top, representative images showing internalized ECAD (green) and DAPI staining (blue). Bar, 20 µm. Bottom, quantification of relative ECAD fluorescence intensity is shown normalized to control. N (fields of view): (I) DMSO=18, CK666=18; (L) DMSO=15, Genz=15; (M) DMSO=19, I3=17 (n=3). N) ECAD internalization was monitored in MCF10A-EV cells subjected to I3 (20 µM, 10 min) treatment, Gal3 KD or Gal3 KD/I3 treatment. N (fields of view): EV, mock=26, I3=27, Gal3 KD=26, Gal3 KD/I3=28 (n=2). p-values in the relevant panels (Unpaired Student’s t-test, two-tailed): ****, <0.0001; ** <0.001, * <0.05

Article Snippet: The following inhibitors of endocytic players were used in this study: CK666 (Arp2/3 complex inhibitor, 50 μM, SML0006 Merck), EIPA (NHE inhibitor, 50 μM; used as macropinocytosis inhibitor, A3085 Merck), I3 (non-permeable Galectin-3 inhibitor, 20 μM, GalectoBiotech), Genz-123346 (ceramide synthase inhibitor, 4 μM, 5.38285 Merck), and (+)-Lactose (pan-galectin inhibitor, 100 mM, L3750 Merck).

Techniques: Control, In Vivo, Fluorescence, Binding Assay, Incubation, Staining, Two Tailed Test