Journal: Cell Death & Disease

Article Title: Loss of RhoA in microglia disables glycolytic adaptation and impairs spinal cord injury recovery through Arhgap25/HIF-1α pathway

doi: 10.1038/s41419-025-07947-9

Figure Lengend Snippet: A Changes of RhoA mRNA expression in injured spinal cord obtained from different GEO databases. B Temporal changes of RhoA protein level in spinal cord following SCI ( n = 5). C Major cell type classification in the GSE196928 dataset. D Distribution map of RhoA expression. E RhoA expression levels across different cell types. F Immunostaining shows the RhoA expression in microglia (IBA-1 + ) at different stages after SCI ( n = 3). Data are presented as mean ± SD. Statistical significance: ∗ p < 0.05, ∗∗ p < 0.005, ∗∗∗ p < 0.001 (vs. sham group).

Article Snippet: RhoA flox/flox and Cx3cr1 Cre mice (provided by Cyagen, Suzhou, China) were intercrossed to obtain RhoA cKO mice with genotype of RhoA flox/flox ; Cx3cr1 Cre , as previously described [ ].

Techniques: Expressing, Immunostaining

Journal: Cell Death & Disease

Article Title: Loss of RhoA in microglia disables glycolytic adaptation and impairs spinal cord injury recovery through Arhgap25/HIF-1α pathway

doi: 10.1038/s41419-025-07947-9

Figure Lengend Snippet: A t-SNE and unsupervised clustering of all cells based on biomarkers. B GSEA analysis highlighting differences in microglial subsets compared to other cell types. C High-resolution reclustering of microglia into ten distinct clusters. D Expression of key markers identified in the reclustered microglia. E Pseudotime trajectory analysis of microglial cells. F Visualization of ten microglial subclusters, each represented by a distinct color. Microglial subsets with significant differences in oxidative phosphorylation pathways ( G subcluster 0, 2, 3, 4, 7) and glycolysis pathways ( H subcluster 2, 3, 4, 7) identified through GSEA analysis. I CellChat analysis showing interactions between glycolysis-differentiated microglia (subcluster 2, 3, 4, 7) and other cell types. J PCA of bulk mRNA transcriptomics in primary microglial cells. K Heatmap illustrating the results of mRNA sequencing analysis. GSEA analysis of glycolysis-related ( L ) and oxidative phosphorylation-related ( M ) differences between flox and cKO groups. N PCA of metabolomic profiling data. O , P Comparative analysis of glycolysis and TCA cycle metabolic changes between cKO and flox groups. RhoA deletion decreases ATP production ( Q ), accompanied by reduced ECAR ( R ), OCR ( S ), and glucose consumption in the culture medium ( T ). Data are presented as mean ± SD; each dot represents an individual mouse. Statistical significance: *** p < 0.001.

Article Snippet: RhoA flox/flox and Cx3cr1 Cre mice (provided by Cyagen, Suzhou, China) were intercrossed to obtain RhoA cKO mice with genotype of RhoA flox/flox ; Cx3cr1 Cre , as previously described [ ].

Techniques: Expressing, Phospho-proteomics, Sequencing

Journal: JACC: Basic to Translational Science

Article Title: Ubiquitin Specific Protease 9X Regulates the Activation of ARK5 and Promotes Progression of Fibrotic Remodeling

doi: 10.1016/j.jacbts.2025.02.014

Figure Lengend Snippet: ARK5 Facilitates TGF-β1–Induced Myofibroblast Transformation via Activating RhoA/MRTF-A Pathway (A) Fibroblasts were infected with USP9X siRNA, then subjected to TGF-β1 stimulation. Total RNA samples were collected and sequenced. The most significantly altered KEGG pathways were displayed. (B) RhoA activity was assessed using western blot analysis after transfecting CFs with ARK5 siRNA or siRNA-control. (C) CFs were infected with siRNA-control, siRNA-USP9X, Ad-GFP, or Ad-ARK5, and then tested for RhoA activity. (D) MRTF-A protein levels in the nuclear and cytosolic fractions were detected by western blot analysis. (E) Immunofluorescence staining was performed to detect the intensity of MRTF-A. Scale bar: 50 μm. (F) CFs were infected with siRNA-control, siRNA-USP9X, Ad-GFP, or Ad-ARK5, and then tested for MRTF-A protein levels. (G) Representative immunofluorescence staining of MRTF-A for each group. Scale bar: 50 μm. n = 3-5, Values represent the mean ± SEM. One-way analysis of variance followed by Tukey’s post hoc test was used for statistical analysis. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. Abbreviations as in , , and .

Article Snippet: Antibodies against USP9X (55054-1-AP), α-SMA (14395-1-AP), mechanosensitive transcription factor myocardin-related transcription factor-A (MRTF-A) (21166-1-AP), Ubiquitin (10201-2-AP), glyceraldehyde 3-phosphate dehydrogenase (60004-1-Ig), PCNA (10205-2-AP), and RhoA (10749-1-AP) were purchased from Proteintech.

Techniques: Transformation Assay, Infection, Activity Assay, Western Blot, Control, Immunofluorescence, Staining

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