Journal: Life Science Alliance

Article Title: Aortic carboxypeptidase-like protein potentiates β1 integrin signaling in mesenchymal progenitors

doi: 10.26508/lsa.202503600

Figure Lengend Snippet: 10T1/2 mesenchymal progenitors were seeded on col1, col1-ACLP, or col1-ACLP+SB hydrogels for 30 min as described in . (A, B, C) Activation of (A) Rac1, (B) RhoA, and (C) Cdc42 was measured using G-LISA activation assays (Cytoskeleton, Inc.). Absorbance was measured at 490 nm. Data represent the mean ± SD from three independent experiments (3 × 10 5 cells per n; n = 3). Statistical comparisons were performed using Welch’s t tests or one-way ANOVA, as appropriate; P < 0.05.

Article Snippet: GTPase activation was analyzed using G-LISA GTPase Activation Assay (BK135, Cytoskeleton) according to the manufacturer’s instructions.

Techniques: Activation Assay

Journal: Clinical and Translational Medicine

Article Title: Mechanism of Gzma‐mediated GEF‐H1 activation in intestinal epithelial cells leading to intestinal barrier dysfunction in sepsis

doi: 10.1002/ctm2.70651

Figure Lengend Snippet: Activation of the GEF‐H1/RhoA signalling axis by Gzma under sepsis conditions. (A) Heatmap of transcriptome sequencing analysis showing differential expression profiles of small GTPases and cytoskeleton‐related genes in the intestinal tissues of WT‐Sham and the CLP sepsis model group (rows: genes; columns: experimental groups; colour scale: relative expression level). (B) Immunofluorescence staining of phalloidin (green, labelling F‐actin) in human colonic epithelial NCM460 cells after co‐culture with LPS‐pretreated NK92MI cells (DAPI staining for nuclei, blue; top row: control group; bottom row: co‐culture group; scale bar = 20 µm). (C) Coomassie blue‐stained SDS‐PAGE gel following Gzma immunoprecipitation (IP); the arrow indicates the GEF‐H1 band. (D) Interaction network between Gzma and candidate binding proteins predicted by the STRING database (Nodes: proteins). (E) Mass spectrum (MS/MS) of high‐affinity peptides binding Gzma to GEF‐H1, showing peptide sequences (EVEGLKDLLVGPGVELLLTPR, LVNLYGLLHGLQAAVAQQDTLMEAR, VGLFAEMTHFQAEEDGGSGMALPTLPR), Xcorr (cross‐correlation coefficient), and charge state. (F) Left: The three‐dimensional structure of the molecular docking of Gzma (yellow) and GEF‐H1 (blue) simulated by the GRAMM software; Right: A close‐up view highlighting the key interacting residues at the interface. (G) SPR analysis of GEF‐H1 binding to Gzma. Sensorgrams show concentration‐dependent binding of Gzma (.125–1 µM). The measured K D is 4.27⨯10 −7 M, indicating a medium‐high affinity interaction. (H) Immunofluorescence co‐localization analysis of GEF‐H1 (red) and Gzma (green) in NCM460 cells after co‐culture with LPS‐pretreated NK cells (DAPI staining for nuclei, blue; top row: control group; bottom row: co‐culture group; scale bar = 20 µm). (I) RhoA G‐LISA activity assay in NCM460 (left) and Caco2 (right) cells after co‐culture with LPS‐stimulated NK cells (mean ± SEM, n = 3; **** p < .0001, one‐way ANOVA). (J) Western blot analysis of GEF‐H1, p‐GEF‐H1 (Ser886), MLC2, p‐MLC2 (Thr18/Ser19), LIMK, p‐LIMK (Thr508), Cofilin, p‐Cofilin (Ser3), and β‐actin (used as a loading control) was performed in NCM460 and Caco2 cells following co‐culture with LPS‐stimulated NK cells.

Article Snippet: RhoA activation levels were quantified using the G‐LISA Assay for Quantifying RhoA Activity (Cytoskeleton, Inc., Denver, CO, USA; Cat. No. BK121), a luminescence‐based detection system.

Techniques: Activation Assay, Sequencing, Quantitative Proteomics, Expressing, Immunofluorescence, Staining, Co-Culture Assay, Control, SDS Page, Immunoprecipitation, Binding Assay, Tandem Mass Spectroscopy, Software, Concentration Assay, Activity Assay, Western Blot

Journal: Clinical and Translational Medicine

Article Title: Mechanism of Gzma‐mediated GEF‐H1 activation in intestinal epithelial cells leading to intestinal barrier dysfunction in sepsis

doi: 10.1002/ctm2.70651

Figure Lengend Snippet: Systematic screening and validation of GEF‐H1 modulator in small molecule compound libraries. (A) Schematic diagram depicting the workflow for screening GEF‐H1 modulator, which includes sequential steps of structure‐based molecular docking, virtual screening, RhoG‐LISA validation, and Western blot verification. The chemical structure of the representative compound Epothilone A is displayed on the right. (B–F) Western blot analysis of p‐GEF‐H1 (S886) and β‐actin expression levels (left panel) in NCM460 cells treated with various microtubule‐targeting compounds, accompanied by corresponding graphs illustrating the percentage inhibition of GEF‐H1 activity (right panel). (G–K) Bar graphs summarizing the fold changes in RhoA activity in NCM460 cells after treatment with different compounds.

Article Snippet: RhoA activation levels were quantified using the G‐LISA Assay for Quantifying RhoA Activity (Cytoskeleton, Inc., Denver, CO, USA; Cat. No. BK121), a luminescence‐based detection system.

Techniques: Biomarker Discovery, Western Blot, Expressing, Inhibition, Activity Assay

Journal: Clinical and Translational Medicine

Article Title: Mechanism of Gzma‐mediated GEF‐H1 activation in intestinal epithelial cells leading to intestinal barrier dysfunction in sepsis

doi: 10.1002/ctm2.70651

Figure Lengend Snippet: Epothilone A maintains intestinal barrier integrity by inhibiting GEF‐H1 to regulate the RhoA/ROCK pathway. Under physiological conditions (homeostasis), GEF‐H1 is maintained in an inactive, phosphorylated state, while RhoA remains bound to GDP. This arrangement keeps downstream effectors, including ROCK, LIMK, MLC2, and Cofilin, in an inactive form; thus, supporting a stable intestinal epithelial cytoskeleton and normal barrier function. However, during sepsis, immune‐cell‐derived Gzma induces the dephosphorylation of GEF‐H1 at Ser886. This activation promotes the formation of RhoA‐GTP, which subsequently activates ROCK. Activated ROCK then phosphorylates LIMK, MLC2, and Cofilin—resulting in cytoskeletal contraction and disruption of tight junctions that ultimately lead to barrier dysfunction. Treatment with Epothilone A stabilizes microtubules and inhibits GEF‐H1 activation; thereby suppressing the RhoA/ROCK pathway and preventing cytoskeletal hypercontraction while restoring epithelial barrier integrity during sepsis.

Article Snippet: RhoA activation levels were quantified using the G‐LISA Assay for Quantifying RhoA Activity (Cytoskeleton, Inc., Denver, CO, USA; Cat. No. BK121), a luminescence‐based detection system.

Techniques: Derivative Assay, De-Phosphorylation Assay, Activation Assay, Disruption

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

Journal: The Journal of Biological Chemistry

Article Title: Claudin-2 suppresses GEF-H1, RHOA, and MRTF, thereby impacting proliferation and profibrotic phenotype of tubular cells

doi: 10.1074/jbc.RA118.006484

Figure Lengend Snippet: Claudin-2 silencing activates RhoA. A–D, WT LLC-PK1 cells (A and D) or MDCK cells (B) or HA-claudin-2 expressing LLC-PK1 cells (C) were transfected with NR or Cldn-1, -2, or -7–specific siRNAs, as indicated. 48 h after transfection, the cells were lysed, and active RhoA was captured using GST–RBD-coupled beads. Precipitated and total RhoA were detected by Western blotting. RhoA in the precipitate (active) is indicated by the arrowhead. Note that in some blots, the antibody also visualized a nonspecific band above the RhoA-specific band. The identity of the RhoA band was verified by silencing. Active RhoA was normalized to the corresponding input (total) RhoA and in each experiment expressed as fold from NR siRNA transfected control. The indicated claudins were detected in total cell lysates to verify silencing. Graphs show means ± S.E. n = 3–5. E, active RhoA staining is elevated in Cldn-2 KO mice. Active RhoA was detected in kidney sections obtained from WT or Cldn-2 KO mice using a RhoA-GTP–specific antibody (left top row). The right top row shows labeling with the secondary antibody alone. Middle row, nuclear stain with DAPI; bottom row, merged images. Bar, 50 μm. Intensity of the labeling was quantified using the Zen software in regions of 100-μm diameter (>30 regions/animals in three KO and two WT animals). Average labeling in samples exposed to the secondary antibody only was subtracted from each intensity value (means ± S.D.)

Article Snippet: PCNA antibody (catalog no. bs-2006R) was from Bioss Antibodies (Woburn, MA), Active RHOA-GTP from NewEast Biosciences (catalog no. 26904) ( 35 ).

Techniques: Expressing, Transfection, Western Blot, Staining, Labeling, Software

Journal: The Journal of Biological Chemistry

Article Title: Claudin-2 suppresses GEF-H1, RHOA, and MRTF, thereby impacting proliferation and profibrotic phenotype of tubular cells

doi: 10.1074/jbc.RA118.006484

Figure Lengend Snippet: A, Cldn-2 silencing activates pMLC through RhoA. LLC-PK1 cells were transfected with NR or claudin-2–specific siRNA with or without RhoA-specific siRNA, and 48 h later the cells were lysed, and phospho-MLC was detected and quantified by Western blotting as in Fig. 2 (means ± S.D., n = 3–6). B, active RhoA is elevated in UUO. The mice were sham-operated or underwent UUO (7 days), as in Fig. 1. Active RhoA was detected in renal tissue sections by immunofluorescent staining with a RhoA-GTP–specific antibody as in Fig. 3. The right images show labeling with the secondary antibody alone. Bar, 50 μm. Intensity of the labeling was quantified using the Zen software in regions of 100-μm diameter (>30 regions/animals in three animals in each group). Average labeling in samples exposed to the secondary antibody only was subtracted from each intensity value (means ± S.D.) C–E, Claudin-2 silencing activates RhoA through GEF-H1. C and D, WT or HA–CLDN-2 expressing LLC-PK1 cells were transfected with NR or Cldn-2–specific siRNA, and 48 h later they were lysed, and active GEFs were precipitated using GST–RHOAG17A. Captured (active) and input (total) GEF-H1 was detected by Western blotting. The input cell lysates were redeveloped with Cldn-2 antibody to verify silencing. Active GEF-H1 values were normalized to the input GEF-H1 (means ± S.D., n = 4). E, WT LLC-PK1 cells were transfected with NR or Cldn-2–specific siRNAs with or without GEF-H1 siRNA, and active RhoA was precipitated and quantified as in Fig. 3 (means ± S.D., n = 4).

Article Snippet: PCNA antibody (catalog no. bs-2006R) was from Bioss Antibodies (Woburn, MA), Active RHOA-GTP from NewEast Biosciences (catalog no. 26904) ( 35 ).

Techniques: Transfection, Western Blot, Staining, Labeling, Software, Expressing

Journal: The FASEB Journal

Article Title: Cdc42 is involved in NC1 peptide–regulated BTB dynamics through actin and microtubule cytoskeletal reorganization

doi: 10.1096/fj.201900991R

Figure Lengend Snippet: Antibodies used in different experiments in this study

Article Snippet: Active RhoA-GTP (AB_1961799) , Mouse , NewEast Biosciences , 26904 , IP , .

Techniques:

Journal: The FASEB Journal

Article Title: Cdc42 is involved in NC1 peptide–regulated BTB dynamics through actin and microtubule cytoskeletal reorganization

doi: 10.1096/fj.201900991R

Figure Lengend Snippet: NC1-peptide–induced Sertoli cell TJ barrier disruption is mediated through Cdc42, which can be blocked by overexpression of Cdc42-T17N, a dominant negative mutant of Cdc42, in Sertoli cell epithelium. A) Regimens used for this study with Sertoli cells cultured in vitro at 0.4 × 106 cells/cm2 that formed an intact epithelium (see Materials and Methods) for different experiments in this report. B) A study by IB that confirmed overexpression of NC1 peptide following its overexpression in Sertoli cells without affecting the steady-state levels of Cdc42 or RhoA. β-Actin served as the protein loading control. Composite data of IB were shown on the right panel, wherein each bar is the mean ± sd of n = 3 experiments. Each experiment had triplicate cultures. **P < 0.01 by ANOVA. C) A pull-down assay was used to assess an activation of Cdc42 (i.e., GTP-bound Cdc42), but not RhoA, following overexpression of NC1 peptide in Sertoli cells with the corresponding negative (−ve) and positive (+ve) controls. N.s., not significantly different. Composite data of n = 3 experiments were shown on the right panel. **P < 0.01 by ANOVA. D) Overexpression of Cdc42-T17N mutant (a dominant negative mutant of Cdc42 by mutating residue Thr17 to Asn17) was confirmed by IB; however, its overexpression alone or cooverexpression with NC1 peptide did not alter the steady-state level of either NC1 peptide or Cdc42. The composite data of n = 3 independent experiments of this study were shown on the right panel. E) Overexpression of Cdc42-T17N mutant was able to eliminate the NC1 peptide–induced Cdc42 activation by quantifying the steady-state level of activated Cdc42 (i.e., Cdc432-GTP) by pull-down assays as noted by IB, including both −ve and +ve controls, with the composite data of n = 3 experiments in the lower panel. **P < 0.01 by ANOVA. F) A study by TER that quantified the Sertoli cell TJ permeability barrier function wherein overexpression of Cdc42-T17N mutant abolished the NC1 peptide–induced Sertoli cell TJ barrier disruption from a representative experiment with n = 4 bicameral units. Three additional experiments using different batches of Sertoli cells yielded similar results. **P < 0.01 by ANOVA by comparing to the other 3 groups. Uncropped blots corresponding to blots in B–E are shown in Supplemental Fig. S1. Ctrl, control.

Article Snippet: Active RhoA-GTP (AB_1961799) , Mouse , NewEast Biosciences , 26904 , IP , .

Techniques: Disruption, Over Expression, Dominant Negative Mutation, Cell Culture, In Vitro, Control, Pull Down Assay, Activation Assay, Mutagenesis, Residue, Permeability