Journal: bioRxiv

Article Title: Vav2 is a master regulator of repair against bacterial pore-forming toxins

doi: 10.1101/2025.07.31.668026

Figure Lengend Snippet: HeLa cells were serum-starved for 30 min, treated with DMSO or 5, 10, or 20 μM (A) Rac inhibitor EHop016, (B) cdc42 inhibitor ML141, (C) Rho inhibitor Y16, (D) ROCK inhibitor Y27632, (E) 20 μM Y16, Y27632, or EHop016 or (F) 20 μM ML141, EHop016, Y16, or all three, and challenged with 31-2000 HU/mL SLO or PFO for 30 min at 37°C. Propidium iodide (PI) uptake was analyzed by flow cytometry. The LC 50 was calculated as described in the methods. Graphs show independent experiments and the mean ±S.E.M, n=5 (A, B, D (SLO), B, C, (PFO)), n=4 (A, D, E, (PFO), D, E, F (SLO)), n=6 (C, SLO), or n=3 (F, PFO). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 denote statistical significance using repeated-measures ANOVA between groups with Tukey post-test.

Article Snippet: Rac inhibitor EHop016 (Cat# S7319), cdc42 inhibitor ML141(Cat# S7686), and FAK/Pyk2b inhibitor PF431396 (Cat# S7644) were from Selleckchem (Houston, TX, USA).

Techniques: Flow Cytometry

Journal: Advanced Science

Article Title: Deterministic Single Cell Encapsulation in Asymmetric Microenvironments to Direct Cell Polarity

doi: 10.1002/advs.202206014

Figure Lengend Snippet: Cdc42 mediates the ability of single MSCs to respond to the asymmetric presentation of integrin ligands. The following inhibitors against small GTPases were tested in gel‐coated MSCs with asymmetric RGD presentation after 1 day in culture in the basal medium: ML141 (10 µM; Cdc42 inhibitor), NSC23766 (NSC; 10 µM; Rac1 inhibitor), Rhosin (30 µM; RhoA inhibitor). DMSO (1:1000 dilution) was used as a negative control. A) Cell volume and sphericity analysis after 2 h drug treatment. i) Cytoplasmic volume, ii) nuclear volume, iii) cell sphericity, and iv) nucleus sphericity. n = 3 independent experiments. * p < 0.05, ** p < 0.01 via nested one‐way ANOVA, followed by Tukey's multiple comparison test. B) Membrane tension analysis with the lipid tension reporter by FLIM after 2 h drug treatment. i) Mean decay lifetime ( τ ) values per cell. τ values across different regions (angles in counterclockwise directions from 0 0 ) of the cell membrane after treatment with ii) DMSO and iii) ML141. n = 12 cells. n.s.: not significant, **** p < 0.0001 via Welch's ANOVA, followed by Dunnett's T3 multiple comparison test. C) Gene expression analysis after 10‐day culture in the medium supplemented with mixed osteogenic and adipogenic cocktails. The inhibitors were added throughout the culture. Osteogenic markers i) Alp , ii) Runx2 , and adipogenic marker iii) Pparg1 . n = 3 independent experiments. * p < 0.05, *** p < 0.001 via ordinary one‐way ANOVA, followed by Tukey's multiple comparison test. All data are shown as mean ± SD.

Article Snippet: The following chemical inhibitors were purchased from Cayman Chemical: Cilengitide (No. 22289), NSC23766 (No. 13196), and ML141 (No. 18496).

Techniques: Negative Control, Comparison, Membrane, Gene Expression, Marker

Journal: Microcirculation (New York, N.Y. : 1994)

Article Title: Cdc42 regulates branching in angiogenic sprouting in vitro

doi: 10.1111/micc.12372

Figure Lengend Snippet: Inhibition of Cdc42 in 3D biomimetic angiogenic model. (A) Schematic of our 3D biomimetic model of angiogenesis. A device is consisted of 2 channels fully embedded inside 2.5mg/ml collagen gel. (B) Cdc42 activity was reduced in half in the presence of 15 μM Cdc42 inhibitor ML141. (C) Representative phase images of sprouts guided by a gradient of angiogenic cocktail including MCP-1, VEGF, PMA, and S1P at Day 4 for control DMSO and Cdc42-inhibited devices. Average invading distance of invading cells into matrix was reduced in the presence of ML141 (N=4 individual experiments); * (p<0.05) indicates statistical significance.

Article Snippet: Methods Using a 3D biomimetic model of angiogenesis in vitro , where endothelial cells were seeded inside a cylindrical channel within collagen gel and sprouted from the channel in response to a defined biochemical gradient of angiogenic factors, we inhibited Cdc42 activity with a small molecule inhibitor ML141 and examined the effects of Cdc42 on the morphogenetic processes of angiogenic sprouting.

Techniques: Inhibition, Activity Assay, Control

Journal: Microcirculation (New York, N.Y. : 1994)

Article Title: Cdc42 regulates branching in angiogenic sprouting in vitro

doi: 10.1111/micc.12372

Figure Lengend Snippet: The effects of Cdc42 on sprout length and density during angiogenesis sprouting. (A) Quantification of sprout density between control DMSO and Cdc42 inhibition conditions. ML141 was initiated at onset of sprouting over a course of 4 days (n=4 individual experiments). The presence of Cdc42 inhibitor slightly decreased the sprout density. (B) Sprout length was quantified at day 4 using images acquired from confocal microcopy. Sprout length was halved when Cdc42 activity was partially inhibited (n=4 individual experiments). (C) Quantification of average sprout angle between DMSO and ML141 devices (n=4 individual experiments) revealed unaltered directional migration of the multicellular sprout structures. (D) Quantification of the number of invading cells demonstrated inhibition of Cdc42 reduced migrating cells into the interstitial matrix (n=4 individual experiments). (E) Representative 3D projections of Z-stack confocal images of sprouts in DMSO and ML141 conditions at day 4. White arrowheads indicate single migrating cells. Scale bar is 100 μm. (F) Quantification of single cell migration among migrating cells in the interstitial matrix revealed a significant increase in the fraction of single migrating cells (n=4 individual experiments). Unit area is 300 μm2. * indicates statistical significance (P<0.05); ns indicates no statistical significance.

Article Snippet: Methods Using a 3D biomimetic model of angiogenesis in vitro , where endothelial cells were seeded inside a cylindrical channel within collagen gel and sprouted from the channel in response to a defined biochemical gradient of angiogenic factors, we inhibited Cdc42 activity with a small molecule inhibitor ML141 and examined the effects of Cdc42 on the morphogenetic processes of angiogenic sprouting.

Techniques: Control, Inhibition, Activity Assay, Migration

Journal: Microcirculation (New York, N.Y. : 1994)

Article Title: Cdc42 regulates branching in angiogenic sprouting in vitro

doi: 10.1111/micc.12372

Figure Lengend Snippet: The effects of antagonizing Cdc42 on branching morphogenesis of angiogenic sprouting. (A) A schematic of two different branching structures (branch and intersegmental branch) observed in angiogenic sprouting in our model guided by a gradient of angiogenic cocktail. (B) Number of branch points is quantified for DMSO vs ML141 conditions. (C) The fraction of sprouts with branches was reduced in the presence of ML141. (D) The fraction of sprouts with intersegmental branches was also reduced when Cdc42 activity was perturbed with ML141. (E) Average length of branch was unaffected by the inhibition of Cdc42. (F) Average length of intersegmental branches was also unaffected by the inhibition of Cdc42. N=4 individual experiments; * (p<0.05) indicates statistical significance; ns indicates no statistical significance.

Article Snippet: Methods Using a 3D biomimetic model of angiogenesis in vitro , where endothelial cells were seeded inside a cylindrical channel within collagen gel and sprouted from the channel in response to a defined biochemical gradient of angiogenic factors, we inhibited Cdc42 activity with a small molecule inhibitor ML141 and examined the effects of Cdc42 on the morphogenetic processes of angiogenic sprouting.

Techniques: Activity Assay, Inhibition

Journal: Microcirculation (New York, N.Y. : 1994)

Article Title: Cdc42 regulates branching in angiogenic sprouting in vitro

doi: 10.1111/micc.12372

Figure Lengend Snippet: Filopodia formation of endothelial cell sprouting upon Cdc42 inhibition. (A) Representative confocal images of phalloidin-stained sprout tip cells showing filopodia-like extensions in DMSO and ML141 conditions. Sprouting was initiated for 3 days. Then 22.5μM ML141 was added for 4hrs before fixation. (B) Average angle of filopodia of sprout tip cells remained unchanged upon inhibition of Cdc42 (n=4 individual experiments). (C) The number of filopodial extensions per sprout tip cells in DMSO and ML141 conditions (n=4 individual experiments) displayed a surge in filopodia-like extension in ML141 treatment. (D) Histogram showing distribution of the filopodia-like extension numbers per sprout tip cells for DMSO and ML141 conditions (n=4 individual experiments). (E) Average length of filopodia-like extensions is quantified for DMSO and ML141 conditions (n=4 individual experiments). (F) Histogram showing distribution of the length of the filopodia-like extensions for DMSO and ML141 conditions. * (p<0.05) and *** (p< 0.001) indicate statistical significance.

Article Snippet: Methods Using a 3D biomimetic model of angiogenesis in vitro , where endothelial cells were seeded inside a cylindrical channel within collagen gel and sprouted from the channel in response to a defined biochemical gradient of angiogenic factors, we inhibited Cdc42 activity with a small molecule inhibitor ML141 and examined the effects of Cdc42 on the morphogenetic processes of angiogenic sprouting.

Techniques: Inhibition, Staining

Journal: NPJ Parkinson's Disease

Article Title: Microglial low-affinity FcγR mediates the phagocytic elimination of dopaminergic neurons in Parkinson’s disease degeneration

doi: 10.1038/s41531-025-01249-9

Figure Lengend Snippet: A CD16/32 accumulates at the leading front of motile microglia. Top panel shows maximum intensity projection of a BV2 cell marked with CD16/32 and DAPI. Leading lamellas (left pole) and the uropod (opposite pole) indicate its direction. Bottom panel shows a 0.5 μm optical plane represented in a scale of 16 colors. Scale on the bottom (white represents the highest value and black the lowest). Leading front lamellas (1) and uropod (2) are indicated (Scale bar: 10 μm). B Illustration of a characteristic motile microglial cell showing essential cytoskeletal elements (Modified from Roig-Martinez et al.) . C Plot of relative fluorescence of motile microglia measured (white broken line) from the image on the left. Note the higher fluorescence at the leading front (1) and a smaller peak at the uropod (2). D BV2 microglia in kinetic shape showing accumulation of Cdc42 at the leading edge (asterisk). E Relative fluorescence of BV2 microglia from panel D indicates the Cdc42 increase at the cell frontal lamella (asterisk) (Scale bar: 10 μm). F Representative image of a BV2 microglial cell expressing a high density of Cdc42 (red) in an F-actin-rich protrusion. The nucleus was counterstained with DAPI (blue). (Scale bar: 3 μm). G Plot profile of relative fluorescence displays high expression of Cdc42 corresponding with high fluorescence of F-actin at the protruding edge (2) compared with the nucleus (1). H Clusters of Cdc42 can be seen at the microglial lamelipodia. Detailed of clusters are shown in higher magnification (yellow arrowheads) from white inset (Scale bar: 10 μm). I Cdc42 is spread as filaments towards to F-actin-rich microglial cell border. Higer magnification from white inset shows detail of the filaments (white arrowheads) (Scale bar: 10 μm). J Higher resolution images from orange inset show that Cdc42 accumulates at the inner part of the F-actin cellular processes (Z confocal steps +3 μm and +4 μm from the culture plate bottom are shown) (Scale bar: 5 μm). K Confocal image of microglial cell establishing a phagocytic cup around a dopaminergic cell (1). The PC12 DA cell is marked with dopaminergic neuronal marker, TH (green), and the microglial cell is stained by F-actin (magenta). (2) Diagram of the engulfing process depicted in 1. (3) Image of F-actin fluorescence intensity of the engulfing microglia seen in 1. Note the higher intensity is displayed at the borders of the phagocytic cup. (4) higher magnification of the phagocytic cup to visualize the characteristic accumulations of F-actin (Scale bar: 10 μm). (5) 3D reconstruction of the phagocytic event displaying the microglia arranging the F-actin-rich phagocytic cup around a dopaminergic cell (yellow arrowhead). L Increasing concentrations of ML141 were safe for BV2 cell viability. MTT cell viability assay for BV2 microglial cells submitted to increasing concentrations of ML141 was performed to test their safety: 500 μg of H 2 O 2 resulted in approximately 50% decrease of the cell viability. But no significant decrease was appreciated after the administration of ML141 or vehicle (*** p ˂0.001 H 2 O 2 significant against every other treatment). M Quantification of the number of PC12 DA cells at 60 min. of interaction with BV2 microglia. BV2 were treated either before or after activation with Cdc42 inhibitor ML141. The proinflammatory-mediated elimination of DA cells was prevented when BV2 cells were incubated with ML141. (Pre-activation inhibition ** p ˂0.01 IFN-γ + LPS vs. ML141/IFN-γ + LPS and ML141. Post-activation inhibition $$$ p ˂0.001 IFN-γ + LPS vs. every condition and **** p ˂0.0001 control vs. IFN-γ + LPS).

Article Snippet: To analyze how the inhibition of the protein Cdc42 affects the preservation of dopaminergic cells, the inhibitor ML141 (Cdc42/Rac1 GTPase Inhibitor, MERCK, Calbiochem, San Diego, CA, USA) was used in BV2/PC12 co-cultures.

Techniques: Modification, Fluorescence, Expressing, Marker, Staining, Viability Assay, Activation Assay, Incubation, Inhibition, Control

Journal: NPJ Parkinson's Disease

Article Title: Microglial low-affinity FcγR mediates the phagocytic elimination of dopaminergic neurons in Parkinson’s disease degeneration

doi: 10.1038/s41531-025-01249-9

Figure Lengend Snippet: A Diagram of the procedure used for the CD16/32 passive immunotherapy. Neuropathological histology was performed in the SNpc (red square). B Representative confocal images showing the SNpc labeled with TH and counterstained with DAPI. Depletion of DA neurons can be appreciated in MPTP-treated animals. However, a protective effect can be seen in mice treated with CD16/32 neutralizing monoclonal antibodies (αCD16/32) (Scale bar: 30 μm). C Quantification of the DA neurons in the SNpc demonstrating a significant decrease in the MPTP group which is prevented by the administration of CD16/32 (*** p ˂0.001 MPTP vs. every treatment except MPTP + Isotype, ### p˂0.001 MPTP + Isotype vs. every treatment except MPTP). D 3D reconstruction of representative microglial cells expressing Iba-1 in different treatments of the experiment (Scale bar: 10 µm). E Quantification of the area of Iba-1 in the SNpc, indicating variation of microglial size when the animals were intoxicated with MPTP. F CD16/32 immunostaining reveals increased immunoreactivity in MPTP-treated animals (Scale bar: 20 μm). G Quantification of CD16/32 cell number shows increased levels in MPTP-treated animals. H Diagram of the procedure used for Cdc42 inhibition. I Representative confocal images of histological analysis. Upper panel: Representative images of TH positive neurons of the SNpc in every treatment (Scale bar: 30 µm). Lower panel: 3D reconstructions illustrating morphology and changes of size in microglial cells expressing Iba-1 (Scale bar: 10 µm). J Quantification of TH positive neurons indicating a significant decrease in the MPTP group and prevention by the previous administration of Cdc42 inhibitor ML141 (** p < 0.01 with respect to saline). K Quantification of the Iba-1 expressing area, which represents changes in microglial size. Microglial activation is present in the MPTP group evidenced by the increased area of Iba-1, which is decreased in the animals intoxicated with MPTP but previously treated with ML141 (*** p < 0.001 with respect to controls, $$ p < 0.01 with respect to ML141). L CD16/32 immunostaining reveals immunoreactivity in the MPTP group, providing a strong inhibition in MPTP-ML141 group (Scale bar: 30 μm). M Quantification of CD16/32+ microglia show a significant increase in the MPTP-treated group (** p < 0.01 compared to ML141 + MPTP).

Article Snippet: To analyze how the inhibition of the protein Cdc42 affects the preservation of dopaminergic cells, the inhibitor ML141 (Cdc42/Rac1 GTPase Inhibitor, MERCK, Calbiochem, San Diego, CA, USA) was used in BV2/PC12 co-cultures.

Techniques: Labeling, Bioprocessing, Expressing, Immunostaining, Inhibition, Saline, Activation Assay