Journal: PLoS ONE

Article Title: Enhanced Neurite Outgrowth of Human Model (NT2) Neurons by Small-Molecule Inhibitors of Rho/ROCK Signaling

doi: 10.1371/journal.pone.0118536

Figure Lengend Snippet: (A) Measurements of neurite outgrowth after 24 h incubation with elevated levels of the Rho activator lysophosphatidic acid (LPA) showed no change in neurite lengths, whereas Ibuprofen increased neurite length (2 independent experiments, 470 to 947 neurites measured). (B) Within 45 min, neurons cultured under control conditions showed a slowly advancing growth cone. (C) Inhibition of RhoA using 500 μM Ibuprofen elicited a weak positive effect on filopodial formation and extension of growth cone. (D) RhoA activation using 100 μM LPA resulted in a growth cone collapse and retraction of the neurite. ***p<0.001 against control by Kruskal-Wallis. Scale bar is 25 μm.

Article Snippet: The RhoA Activation Assay Biochem Kit (bead pull-down format) was purchased from Cytoskeleton Inc. (Denver, CO, USA).

Techniques: Incubation, Cell Culture, Control, Inhibition, Activation Assay

Journal: PLoS ONE

Article Title: Enhanced Neurite Outgrowth of Human Model (NT2) Neurons by Small-Molecule Inhibitors of Rho/ROCK Signaling

doi: 10.1371/journal.pone.0118536

Figure Lengend Snippet: (A) A RhoA bead pull down assay revealed lowered levels of activated RhoA in Ibuprofen treated cells. Treatment with downstream Rho kinase inhibitor Y-27632 showed no difference in RhoA level compared to control. After treatment with lysophosphatidic acid, significantly more phosphorylated RhoA could be detected in the Western Blot. Treatment with downstream Rho kinase inhibitor Fasudil showed no difference in RhoA level compared to control. Cell lysates were blotted and stained with RhoA-specific monoclonal antibody. Staining is visualized with horseradish peroxidase and the 3, 3′-diaminobenzidine substrate. Molecular weight of stained protein bands corresponds to about 20 kDa as reported for RhoA. (B) Relative RhoA level was decreased to approximately 50% of control after Ibuprofen treatment whereas RhoA level after LPA treatment was increased to 200% of control. Data are normalized grey values ± SEM of 4 independent experiments.

Article Snippet: The RhoA Activation Assay Biochem Kit (bead pull-down format) was purchased from Cytoskeleton Inc. (Denver, CO, USA).

Techniques: Pull Down Assay, Control, Western Blot, Staining, Molecular Weight

Journal: PLoS ONE

Article Title: CD148 Tyrosine Phosphatase Promotes Cadherin Cell Adhesion

doi: 10.1371/journal.pone.0112753

Figure Lengend Snippet: A) Effects of CD148 in E-cadherin contact formation were examined by a calcium-switch assay and immunofluorescence staining. CD148 WT, but not CS, promotes E-cadherin contact formation in A431D/E-cadherin WT cells, as evidenced by the expanded and intense E-cadherin distribution (left panels), while CD148 WT shows no effects in A431D/E-cadherin 764 AAA cells (right panels). B) Activities of Rac1, Cdc42, and RhoA were measured in the condition of calcium switch assay. Active and total levels of Rac1, Cdc42, and RhoA proteins were assessed by the pull-down assays and/or immunoblotting described in the “ ” (left panels). The activity was normalized to total amount of protein using densitometry and expressed as fold change (right panels). The data show means ± SEM of quadruplicate determinations. **P<0.05 vs. CD148 (−) cells.

Article Snippet: RhoA, Rac1, and Cdc42 activities were assessed using the Rac1 and RhoA pull-down activation assay kit (Cytoskeleton, Inc., Denver, CO) according to the manufacturer’s instruction.

Techniques: Immunofluorescence, Staining, Western Blot, Activity Assay

Journal: PLoS ONE

Article Title: CD148 Tyrosine Phosphatase Promotes Cadherin Cell Adhesion

doi: 10.1371/journal.pone.0112753

Figure Lengend Snippet: A and B) CD148-introduced or CD148-negative A431D/E-cadherin WT (panel A) and A431D/E-cadherin 764 AAA (panel B) cells were subjected to a hanging drop assay. Rac1, Cdc42, and RhoA activities were assessed at the indicated time points. Active and total levels of Rac1, Cdc42, and RhoA proteins were assessed by pull-down assays and/or immunoblot analysis (left panels). The relative levels of active versus total Rac1, Cdc42, and RhoA were quantified by densitometric analysis (right panels). The data show means ± SEM of quadruplicate determinations. **P<0.05 vs. CD148 (−) cells. CD148 WT, but not CS, increases Rac1 activity in A431D/E-cadherin WT cells, while this effect is not observed in A431D/E-cadherin 764 AAA cells. C) Effects of CD148 WT in cell-cell adhesion were assessed by a hanging drop assay in the presence or absence of Rac1 inhibitor NSC 23766 (75 µM). CD148 increase of cell-cell adhesion is largely diminished by Rac1 inhibition.

Article Snippet: RhoA, Rac1, and Cdc42 activities were assessed using the Rac1 and RhoA pull-down activation assay kit (Cytoskeleton, Inc., Denver, CO) according to the manufacturer’s instruction.

Techniques: Western Blot, Activity Assay, Inhibition

Journal: Virulence

Article Title: Integrin β3 α2,6-sialylation by the sialyltransferase ST6Gal1 promotes human cytomegalovirus entry into fibroblasts

doi: 10.1080/21505594.2026.2732519

Figure Lengend Snippet: Integrin β3 sialylation participates in HCMV-induced cytoskeleton rearrangement during viral entry. (A) Integrin β3 sialylation defect resulted in downregulation of the Integrin β3-src signaling pathway. (B) The sialylation defect also resulted in impaired HCMV-induced RhoA inactivation, as shown by quantification. Data are presented as mean ± standard deviation ( n = 3), with **adjusted p < 0.01 vs The ST6Gal1 shRNA group, ***adjusted p < 0.001 vs The ST6Gal1 shRNA group. (C) Integrin β3 S85 sialylation was found to facilitate stress fiber disassembly and promote viral nuclear translocation. Representative immunofluorescence images for each group are shown (magnification: 600×, scale bar: 25 μm).

Article Snippet: Intracellular GTP-RhoA activity was determined using the G-LISA RhoA Activation Assay Biochem Kit (Cytoskeleton Inc., Cat#BK124, Denver, USA) according to the manufacturer’s instructions.

Techniques: Standard Deviation, shRNA, Translocation Assay, Immunofluorescence

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: Frontiers in Molecular Biosciences

Article Title: RhoA/ROCK-YAP/TAZ Axis Regulates the Fibrotic Activity in Dexamethasone-Treated Human Trabecular Meshwork Cells

doi: 10.3389/fmolb.2021.728932

Figure Lengend Snippet: RhoAG14V promoted the expression of Collagen I and α-SMA and regulated YAP/TAZ in HTM cells (A) The bands of RhoA, α-SMA, and Collagen I proteins (B) Quantification analyses of RhoA, α-SMA, and Collagen I expression levels (C) RT-qPCR analyses of α-SMA and Collagen I mRNAs (D) The bands of YAP/TAZ, pYAP, pTAZ, and CTGF proteins (E) Quantification analyses of YAP, TAZ, pYAP, pTAZ, and CTGF proteins (F) RT-qPCR analyses of YAP, TAZ, and CTGF mRNAs (G) Representative fluorescent graphs of YAP/TAZ labeled by specific antibody and (H) quantitative analysis of YAP/TAZ in the nucleus. Scale bar, 50 μm; * p < 0.05; ** p < 0.01.

Article Snippet: The constitutively active RhoA (RhoAG14V) was provided by Beijing Syngentech Co., Ltd.

Techniques: Expressing, Quantitative RT-PCR, Labeling

Journal: bioRxiv

Article Title: Astrocytes close the critical period for visual plasticity

doi: 10.1101/2020.09.30.321497

Figure Lengend Snippet: (a) Top, volcano plot analysis representing the 2096 quantified proteins with at least 3 total peptides in all replicates enriched with biotinylated WFA lectin in KD and WT mice. Binding partners were obtained by using quantitative label-free mass spectrometry analysis performed from three replicates. Dashed vertical lines denote absolute fold change of 1.5 and the dashed horizontal line denotes the adjusted P-value of ratio significance of 0.05. Selected enriched proteins in WT (green) and KD (purple) samples are shown. Proteins from the enriched pathways Rho-GTPase activate KTN1 (blue) and Rho-GTPase activate ROCK (red) are highlighted. External plots show proteins with peptides identified only in one sample type (left in KD and right in WT). Bottom, volcano plot analysis representing interactors of Cx30 from the Rho-GTPase activate KTN1 (blue) and Rho-GTPase activate ROCK (red) pathways. The fold-change in WT (n=5) versus KD mice (n=4) are shown with selected proteins (Rock2 and Myosin 14) quantified with an absolute fold change ≥ 1.5, an adjusted P-value ≤ 0.05 and with ≥ 3 peptides. (b) Immunostaining for RhoA-GTP showed a marked increase in KD (n=27, P=0.0003), KD MD (n=16, P<0.0001) and WT MD mice (n=16, P<0.0001) compared to WT mice (n=22) (slices from 3 mice per group) (Kruskal-Wallis (KW=41.23) followed by Dunn’s post-hoc test. (c) Immunostaining for MMP9 shows a marked increase in KD mice (n=28, P<0.0001, DF=71), KD MD (n=13, P=0.0059, DF=71) and MD mice (n=12, P=0.0322, DF=71) compared to WT mice (n=22) (ANOVA (F(3,71)=15.07) followed by Tukey’s post-hoc test). (d) Diagram depicting the protocol for experimental treatment with Fasudil. (e) Fasudil rescued MMP9 levels in KD mice (KD+Fasudil: n=25 from 5 mice, KD=28 slices from 3 mice, P<0.0001), while it had no effect in WT mice (WT+Fasudil: n=34 from 6 mice, WT: 22 slices from 3 mice, P>0.9999, Kruskall-Wallis (KW=55.98) followed by Dunn’s post-hoc test). (f) Fasudil rescued perineuronal nets levels in KD mice (KD+Fasudil: n=52 from 5 mice; KD, n=177 from 16 mice, P<0.0001) while it had no effect in WT mice (WT+Fasudil, n=129 from 6 mice, WT, n=226 from 16 mice, P=0.1510) (Kruskall-Wallis (KW=71.65) followed by Dunn’s post-hoc test).

Article Snippet: The following primary antibodies were used: Cx30 rabbit polyclonal (1:500, 71-2200, Zymed), Cx43 mouse monoclonal (1:500, 610061 BD Biosciences), GFAP mouse monoclonal (1:500, G3893, Sigma-Aldrich), chick anti GFP (1:500, AB13970, Abcam), Parvalbumin mouse monoclonal (1:500, 235, SWANT), MMP9 rabbit (1:500, 3852, cell signaling), anti-active RhoA mouse monoclonal (1:500, 26904, NewEast Biosciences), Lectin from Wisteria Floribunda, biotin conjugate (1:500, L1516-2MG, Sigma-Aldrich).

Techniques: Binding Assay, Mass Spectrometry, Immunostaining