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: Cell reports

Article Title: The actin and microtubule network regulator WHAMM is identified as a key kidney disease risk gene

doi: 10.1016/j.celrep.2025.115462

Figure Lengend Snippet: (A) Percent of cytotoxicity of primary tubule cells (TECs) treated with PBS or cisplatin in the presence or absence of CK666 (CK) or latrunculin A (LatA). (B) Left: immunoblots of cleaved caspase-3 (C-CASP3), full-length gasdemrin D (F-GSDMD), cleaved GSDMD (N-GSDMD) protein, and GAPDH in TECs treated with PBS, cisplatin, or CK666 plus cisplatin. Right: quantification of immunoblots normalized to relative GAPDH level. (C) Left: confocal imaging of F-actin and ASC in Whamm +/+ and Whamm −/− TECs treated with cisplatin, and in Whamm +/+ TECs treated with CK666 plus cisplatin. Scale bars, 20 μm. Right: ImageJ analysis for the colocalized region (arrow): (upper) Whamm +/+, (middle) Whamm −/−, (lower) Whamm +/+ with CK666. (D) Quantification of ASC specks in TECs treated with cisplatin or CK666 plus cisplatin per region of interest (ROI). (E) WHAMM transcript levels in human renal proximal tubular epithelial cells (RPTECs) transduced with sgWHAMM or non-target (sgNST) virus. (F) Immunoblot of WHAMM in RPTECs after CRISPRi-mediated WHAMM silencing. (G) Quantification of WHAMM immunoblot normalized to GAPDH level. (H) F-Actin staining in RPTECs after CRISPRi-mediated silencing of WHAMM. Scale bar, 20 μm. Box indicates zoomed area. (I) ImageJ analysis of F-actin density and actin branch features. (J) WHAMM transcript levels in RPTECs following CRISPRi-mediated WHAMM silencing and treated with 40 μM of cisplatin for 24 h. (K) Percent of cytotoxicity in RPTECs after sg WHAMM or sgNST infection and treated with cisplatin for 24 h. (L) Transcript levels of BAX , CASPASE3 (CASP3) , CASP9 , and CASP1 , interleukin 1β ( IL-1B ), and IL-18 in RPTECs after sg WHAMM or sgNST infection and treated with cisplatin for 24 h. Data are presented mean ± SEM. p values were determined by one-way ANOVA or unpaired t test in GraphPad Prism 10 software. * p < 0.05, *** p < 0.001, **** p < 0.0001. See also .

Article Snippet: CK666 (#29038, Cayman) injections were continued for the rest of the two days of cisplatin-AKI and sacrificed on day 3 rd .

Techniques: Western Blot, Imaging, Transduction, Virus, Staining, Infection, Software

Journal: Cell reports

Article Title: The actin and microtubule network regulator WHAMM is identified as a key kidney disease risk gene

doi: 10.1016/j.celrep.2025.115462

Figure Lengend Snippet: (A) Experimental scheme of generating the CK666 (CK)-cisplatin model (CK-Cis). (B) Serum creatinine (sCr) and blood urea nitrogen (BUN) in the CK-Cis model ( n = 3–6 mice per group). (C) Transcript levels of Havcr1 , and Lcn2 in the CK-Cis model. (D) Whamm transcript level in the CK-Cis model. (E) H&E and PAS staining in kidney sections from the CK-Cis model. Scale bar, 50 μm. (F) The proposed mechanism for WHAMM in kidney disease. (G) Transcript levels of Nlrp3 , IL-1β , and IL-18 in the CK-Cis model or mice fed on adenine or control diet (CPD) (n = 4–5 mice per group). (H) Immunoblots of NLRP3, CASP1, cleaved CASP1 (P20), GSDMD-F, GSDMD-N, and GSDME in the CK-Cis model. (I) Immunoblots for cleaved GSDME and GAPDH in kidneys of control ( n = 3) or adenine-fed mice ( n = 6) injected with CK666 ( n = 5). (J) Quantification of NLRP3, CASP1, cleaved CASP1 (P20), GSDMD-F, GSDMD-N, and cleaved GSDME immunoblots in the CK-Cis model. Data are presented mean ± SEM. p values were determined by one-way ANOVA in GraphPad Prism 10 software. * p < 0.05, *** p < 0.001, **** p < 0.0001. See also .

Article Snippet: CK666 (#29038, Cayman) injections were continued for the rest of the two days of cisplatin-AKI and sacrificed on day 3 rd .

Techniques: Staining, Control, Western Blot, Injection, Software

Journal: bioRxiv

Article Title: Spatial distribution of cytoskeleton-mediated feedback controls cell polarization: a computational study

doi: 10.1101/2025.04.12.648264

Figure Lengend Snippet: (A-D) Differentiated neutrophils with F-actin-rich fronts (denoted by LifeAct-miRFP703) were imaged over time, and their shape and displacement were measured. Corresponding perturbations were simulated using Model 1 (local inhibition) and Model 2 (global inhibition), with the first 400 seconds showing STEN activity before effector recruitment (A–B) or drug addition (C–D), and the last 500 seconds showing STEN activity post-effector recruitment (A– B) or drug addition (C–D). The three colors in the kymograph represent Ras (Red), PKB (Blue), and PIP2 (Green). A. A 488 nm laser was switched on at frame 50 to globally recruit cytosolic CRY2PHR-mCherry-KRas4B G12V ΔCAAX to the cell periphery, increasing Ras activity and activating the STEN network. B. A 488 nm laser was switched on at frame 47 to globally recruit cytosolic CRY2PHR-mCherry-INP54P to the cell periphery, reducing PIP2 levels, which serves as the back molecule in STEN in both models. C. Differentiated neutrophils were pre-treated with 50 µ M CK666 to inhibit branched actin (denoted by LifeAct-miRFP703), turning off its positive feedback. D. Cells were pre-treated with 5 µ M Latrunculin B to completely remove actin polymerization (denoted by LifeAct-miRFP703), turning off both positive and negative feedback. Scale bar: 5 µ m.

Article Snippet: 10 μ M Latrunculin B (Sigma-Aldrich; Cat #428020) or 50 mM CK666 (EMD Millipore; Cat #182515) stock was prepared in dimethyl sulfoxide (DMSO, Sigma-Aldrich; Cat #D2650).

Techniques: Inhibition, Activity Assay

Journal: The Journal of Cell Biology

Article Title: Rectification of planar orientation angle switches behavior and replenishes contractile junctions

doi: 10.1083/jcb.202309069

Figure Lengend Snippet: Drug treatment reagents and concentrations

Article Snippet: CK666 (Cat#3950; TOCRIS) , Arp2/3 inhibitor , DMSO diluted 1:20 in H 2 O , 250 μm.

Techniques: Concentration Assay