Journal: eLife

Article Title: Global molecular landscape of early MASLD progression in human obesity

doi: 10.7554/eLife.109534

Figure Lengend Snippet: ( A ) Workflow of the LX2 experiment in panels A–F ( n = 8–10). ( B ) GTPase inhibitors NSC23766 (Rac1 inhibitor) and ML141 (Cdc42 inhibitor) significantly reduced pro-collagen secretion from HSC-like LX-2 cells and ( C ) decreased gene expression of COL1A1 and COL1A2 under basal conditions. ( D ) TGF-β administration increased pro-collagen secretion by 32% and ( E ) doubled collagen gene expression in LX2 cells. ( F ) NSC23766-mediated GTPase inhibition impairs pro-collagen secretion from LX-2 cells after TGF-β treatment. Pro-collagen secretion was determined by ELISA and gene expression levels were assessed by quantitative PCR (qPCR). Asterisks (*) denote p-value <0.05 for statistical significance from one-way ANOVA with Holm–Sidak multiple comparisons (B, C), unpaired t -test (D, E), Kruskal–Wallis Test with Dunn's multiple comparisons ( F ). ( G ) Gene expression of selected GTPases in LX-2 cells with and without Elafibranor ( n = 3).

Article Snippet: Chemical compound, drug , ML141 , MedChemExpress , Cat. #: HY-12755 , .

Techniques: Gene Expression, Inhibition, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: Nature cell biology

Article Title: An Apical MRCK-driven Morphogenetic Pathway Controls Epithelial Polarity

doi: 10.1038/ncb3592

Figure Lengend Snippet: ( a,b ) FRET analysis of Cdc42 activity at the apical aspect of MDCK cells conditionally expressing Dbl3-myc. ( c,d ) Confocal microscopy of pMLC and F-actin at the apical membrane domain of MDCK cells conditionally expressing Dbl3-myc induced with tetracycline in the absence or presence of Cdc42 inhibitor. White arrowheads highlight the apical membrane cortex labelled for F-actin. ( e,f ) Analysis of aPKCζ localization in MDCK cells with tetracycline-inducible Dbl3-myc expression. White arrowheads point to the lateral junctional complex. ( g ) Schematic diagram illustrating activation of two effector mechanisms, MRCK/Myosin-II and Par6-aPKC by Dbl3 stimulated apical Cd42-activation. Quantifications show shown are (b) box blots (25th to 75th percentiles, with a line at the median; whiskers extend to the max/min data points; n=36 cells -Tet and n=46 cells +Tet; p value was calculated with a Wilcoxon test), or (d, f) based on n=3 independent experiments and showing the data points, means ± 1 SD (in black), the total number of cells analysed for each type of sample across all experiments, and p-values derived from t-tests. Scale bars: 10 μm.

Article Snippet: Blebbistatin (10μM final concentration), the Cdc42 inhibitor ML141 (10μM), and the ROCKI/II inhibitor GSK 269962 (10nM) were purchased from Tocris Bioscience.

Techniques: Activity Assay, Expressing, Confocal Microscopy, Membrane, Activation Assay, Derivative Assay

Journal: Nature cell biology

Article Title: An Apical MRCK-driven Morphogenetic Pathway Controls Epithelial Polarity

doi: 10.1038/ncb3592

Figure Lengend Snippet: ( a,b ) Levels of EGFP-Lifeact (EGFP-LA), Par polarity and brush border proteins localized at the apical membrane domain in MDCK cells conditionally expressing Dbl3-myc, treated with blebbistatin and followed by washout (WO). ( c,d ) Levels of EGFP-Lifeact (EGFP-LA) enrichment at the apical membrane domain of MDCK cells conditionally expressing Dbl3-myc following blebbistatin treatment and washout in the absence or presence of BDP5290 to inhibit catalytic activity of MRCK. ( e-i ) Measurements of co-localization coefficients of PAR polarity and brush border proteins at the apical membrane domain in MDCK cells conditionally expressing Dbl3-myc transiently inhibited with blebbistatin followed by wash out for 2 hours (green indicates co-localisation, see also and ; arrowheads point to apical membrane). ( j ) Schematic model of polarity induced by apical stimulation of Cdc42, activating a dual effector mechanism to generate asymmetric actomyosin contractions, apical PAR domain formation and membrane morphogenesis. Bright green represents co-localization of Par and brush border proteins. ( k-n ) Quantification of confocal sections of pupal retinas stained for the apical markers aPKC and Moesin comparing wild type cells and gek mutant cells ( k, l ), or wild type cells and cells mutant for gek expressing Sqh EE ( m, n ). Quantifications in panels b, d, f, h, and i are based on n=3 independent experiments and shown are the data points, means ± 1 SD (in black), the total number of cells analysed for each type of sample across all experiments, and p-values derived from t-tests. Quantifications in panels k-n of protein expression levels compare measurements from wild type and neighbouring mutant cells (paired within sections; see for representative images); k, n=10 animals; l, n=8 animals; m and n, n=7 animals; p values were calculated with t-tests). Scale bars: 10 μm.

Article Snippet: Blebbistatin (10μM final concentration), the Cdc42 inhibitor ML141 (10μM), and the ROCKI/II inhibitor GSK 269962 (10nM) were purchased from Tocris Bioscience.

Techniques: Membrane, Expressing, Activity Assay, Staining, Mutagenesis, Derivative Assay

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: 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