Journal: Cytoskeleton (Hoboken, N.j.)

Article Title: ARC Expands the DAAM1 Microexon‐Mediated Actin– RHOA / ROCK Interplay

doi: 10.1002/cm.22051

Figure Lengend Snippet: ARC protein expands the feedback loop between Actin, RHOA/ROCK signaling cascade, and the DAAM1 microexon. (a) Representative immunocytochemistry assay images of mature glutamatergic neurons (day in vitro 21; DIV21), stained with neuronal morphology marker β3‐Tubulin (green) and IEG‐positive nuclei for ARC (magenta), cFOS (red), and EGR1 (cyan). (b) Percentage quantification of ARC, cFOS, and EGR1‐positive neuronal nuclei normalized to their total number based on DAPI (not shown). One dot represents one cell line average, for which 3–4 randomly selected regions of interest were analyzed. Different point shapes represent biological replicates. p values from a two‐way ANOVA test with replicate and genotype as factors. (c) Representative images of ARC immunohistochemistry (green) and DAPI‐stained nuclei (blue) in sections of the dentate gyrus of the hippocampus from PD21 mice. (d,e) Quantifications of IEG‐positive nuclei in the hippocampal dentate gyrus across images normalized to the dentate gyrus size for ARC in basal conditions (d), or 1.5 h after learning (e) with ARC (left panel) and cFOS (right panel). p values from two‐sided Wilcoxon rank‐sum tests. One dot represents one animal for which 3–6 coronal views of the hippocampus were analyzed. (f) Overview of the phenotypes observed following the removal of Daam1‐MIC and their connection to ARC and the RHOA/ROCK signaling pathway (Habas et al. ; Liu et al. ; Schönichen and Geyer ). DVL interacts with DAAM1, which removes its autoinhibition. This is further enhanced by additional interactions with RHOA. Active DAAM1 triggers RHOA activation via an unclear mechanism linked to DAAM1's C‐terminal region (dashed line) and does not rely on direct binding between RHOA and DAAM1. Microexon removal reduces RHOA binding to DAAM1's N‐terminal region. This is proposed to lead to reduced hydrolysis of RhoA‐GTP, which increases the pool of active RHOA and, in turn, hyperactivates the RHOA/ROCK signaling cascade. Impaired actin cytoskeleton presumably enhances local translation of ARC mRNA and increases ARC protein levels. Arrows show the direction of the event. The dotted arrow suggests a potential, unknown feedback loop involving actin polymerization, ARC levels, and RHOA/ROCK pathway activation. ARC, Activity‐regulated cytoskeleton‐associated protein; GAP, GTPase‐activating protein; GEF, Guanine nucleotide exchange factor; GTP, Guanosine triphosphate; GDP, Guanosine diphosphate.

Article Snippet: “ ARC Expands the DAAM1 Microexon‐Mediated Actin– RHOA / ROCK Interplay .” Cytoskeleton 83 , no. 3 : 123 – 127 .

Techniques: Immunocytochemistry, In Vitro, Staining, Marker, Immunohistochemistry, Activation Assay, Binding Assay, Activity Assay

Journal: Cell Reports Medicine

Article Title: A Rac-specific competitive inhibitor of guanine nucleotide binding reduces metastasis in triple-negative breast cancer

doi: 10.1016/j.xcrm.2025.102233

Figure Lengend Snippet: A41 selectively impairs RAC protein activation (A) Representative surface plasmon resonance (SPR) sensograms of binding of immobilized RHOA or CDC42 with indicated increasing concentrations of A41 ( N > 3). (B) Effect of A41 on GEF-stimulated RAC2, RHOG, RHOA, and CDC42 nucleotide exchange. Purified small G proteins were pre-loaded with GDP and then nucleotide exchange was monitored by the increase in fluorescence following mant-GTP binding in the absence and presence of A41 (5 μM) ( N = 3). K obs is expressed as mean ± SEM. ∗ p < 0.001 vs. control. (C) Effect of A41 on RAC1 G30S activation. RAC1 G30S activation was monitored by the increase in fluorescence following mant-GTP binding in the absence and presence of A41 (5 μM). The GEF TRIO was used to induce nucleotide exchange ( N = 3). Data are expressed as mean ± SEM. (D) Immunoblot analysis and associated quantification of RAC-GTP level and total RAC expression in NIH-3T3 fibroblasts expressing RAC wild-type (RAC1 WT) or RAC1 oncomutants (RAC1 P29S and RAC1b) in the absence (Ctrl) and presence of A41 (10 μM). RAC activation was measured as the ratio of RAC-GTP to total RAC and expressed relative to Ctrl condition. Data are presented as mean ± SEM. ∗∗∗ p < 0.001 vs. controls.

Article Snippet: RAC1, RHOA and CDC42 purified proteins (respectively RH01, RC01 and CD01, Cytoskeleton) were diluted to 5 μg/mL in Na + acetate buffer (pH 5.0) and injected into sensor chip CM5 (GE Healthcare) in a Biacore T200 (GE Healthcare) that was activated with NHS/EDC buffer.

Techniques: Activation Assay, SPR Assay, Binding Assay, Purification, Fluorescence, Control, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Modulation of Phosducin-Like Protein 3 (PhLP3) Levels Promotes Cytoskeletal Remodelling in a MAPK and RhoA-Dependent Manner

doi: 10.1371/journal.pone.0028271

Figure Lengend Snippet: A commercially available RhoA-GTP/Rhotekin pull down assay was carried out on siRNA silenced PhLP3 LB01 cells. Samples were collected 24 h, 48 h and 72 h post transfection and activated RhoA was detected at 72 h post-knockdown with an anti-Rho A antibody but not in plasmid only control cells (A). LB01 cells transfected as in panel A for 72 h were then treated for 24 h with the ROCK inhibitor Y2763 (either 3 µM or 10 µM), fixed and stained for actin (rhodamine phalloidin, red). F-actin disassembly was observed at both Y2763 concentrations and the concentration of peripheral actin was observed with 10 µM Y2763 (B).

Article Snippet: Activation of RhoA was determined using a commercially available pull down assay based upon detecting the interaction of the active GTP-bound RhoA with the Rho-binding domain (RBD) of Rhotekin (RhoA Activation Assay Kit, STA-403-A, Cell Biolabs Inc, USA).

Techniques: Pull Down Assay, Transfection, Knockdown, Plasmid Preparation, Control, Staining, Concentration Assay

Journal: Acta Biochimica et Biophysica Sinica

Article Title: LINC00891 regulated by miR-128-3p/GATA2 axis impedes lung cancer cell proliferation, invasion and EMT by inhibiting RhoA pathway

doi: 10.3724/abbs.2022005

Figure Lengend Snippet: LINC00891 regulates A549 and H460 cell functions via the RhoA pathway (A) A549 and H460 cells were transfected with LINC00891 overexpression vector, LINC00891 siRNA or respective negative controls, and the activated RhoA was determined using RhoA Activation Assay Kit. (B) Cells were treated with 15 μM CCG-1423 for 24 h, and the activated RhoA was assessed using RhoA Activation Assay Kit. Cells were treated with LINC00891 siRNA alone or together with 15 μM CCG-1423, and cell proliferation (C), invasion (D), and migration (E) were analyzed. (F) Western blot analysis was used to measure the protein levels of E-cadherin, Vimentin, Snail and Slug. n=5 in each group, *P<0.05, **P<0.01.

Article Snippet: RhoA activity was assessed using RhoA Activation Assay Kit (Merck Millipore, Billerica, USA) according to the manufacturer’s instructions.

Techniques: Transfection, Over Expression, Plasmid Preparation, Activation Assay, Migration, Western Blot