Journal: Bioactive Materials

Article Title: Mechanically sensitized hydrogel microspheres trigger membrane receptor switch for cartilage repair

doi: 10.1016/j.bioactmat.2026.03.017

Figure Lengend Snippet: Effect of OBNC microspheres on regulating MSCs. A) The expression of FAK and p-FAK detected by Western blot analysis. B) Quantitative analysis of the Western blot results. C) The expression of the integrin-α5 gene. D) The expression of the integrin-α8 gene. E) The expression of the Lamb-2 gene. F) The expression of the Spp-1 gene. G) The expression of the Vav-3 gene. H) The expression of the CDC-42 gene. I) Detection of the CDC-42 active protein by a GLISA kit. J) Detection of Rac-1 active protein by a GLISA kit. K) Detection of Rho A active protein by a GLISA kit. L) Mechanism of OBNC hydrogels triggering the membrane receptor switch and activating integrin receptors. (ns: non-significant, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001).

Article Snippet: RhoA GLISA Activation Assay (No. BK124, Cytoskeleton, USA), CDC42 GLISA Activation Assay (No. BK127, Cytoskeleton, USA), and Rac1 GLISA Activation Assay (No. BK128, Cytoskeleton, USA) were used to measure the activation of the GTPase protein family.

Techniques: Expressing, Western Blot, Membrane

Journal: STAR Protocols

Article Title: Protocol to identify covalent inhibitors targeting RhoA Cys16

doi: 10.1016/j.xpro.2026.104494

Figure Lengend Snippet: Identification of CL16 binding to RhoA by gel-based ABPP experiment (A) Rationale of gel-based ABPP for screening compound binding to RhoA in vitro . Purified RhoA protein was pre-treated with solvent control or library compounds followed by labelling with fluorescent activity-based probes. (B) Expected outcomes in gel-based ABPP. In the 1 st round of screening, compound binding to RhoA results in a decrease in in-gel fluorescence (FL) intensity. Further validation of the binding can be performed by experiments using varying concentrations of the hit compounds. In the 2 nd round of screening, selected hits are examined for their binding to Cdc42 and Rac1 to determine their selectivity for RhoA.

Article Snippet: RhoA Pull-down Activation Assay Biochem Kit (bead pull-down format) , Cytoskeleton , Cat# BK036.

Techniques: Binding Assay, In Vitro, Purification, Solvent, Control, Activity Assay, Fluorescence, Biomarker Discovery

Journal: STAR Protocols

Article Title: Protocol to identify covalent inhibitors targeting RhoA Cys16

doi: 10.1016/j.xpro.2026.104494

Figure Lengend Snippet: Validation of CL16-RhoA binding by LC-MS/MS experiments (A) Workflow of LC-MS/MS experiments to detect RhoA-CL16 engagement in HCT116 cells. CL16-treated cells were harvested, digested and analyzed by LC-MS/MS. (B) Representative MS/MS showing CL16 modification on RhoA Cys16 in HCT116 cells. (C) Workflow of LC-MS/MS experiments using CL16-alkyne (a CL16-molecular probe) to study target profile of CL16. (D) Volcano plot revealing protein targets of CL16 identified by CL16-molecular probe. Statistical analyses were performed by two-tailed Student’s t-test by MS Excel. (E) Venn diagram summarizing the protein targets of CL16 identified in (A) and (C), highlighting RhoA as the primary target.

Article Snippet: RhoA Pull-down Activation Assay Biochem Kit (bead pull-down format) , Cytoskeleton , Cat# BK036.

Techniques: Biomarker Discovery, Binding Assay, Liquid Chromatography with Mass Spectroscopy, Tandem Mass Spectroscopy, Modification, Two Tailed Test

Journal: STAR Protocols

Article Title: Protocol to identify covalent inhibitors targeting RhoA Cys16

doi: 10.1016/j.xpro.2026.104494

Figure Lengend Snippet: Biochemical assays to Investigate RhoA inhibition by CL16 (A) Expected results of RhoGTPase activity assay in which CL16 selectively impairs the activation of RhoA and not Rac1 and Cdc42. (B) Schematic diagram illustrating the workflow of the co-immunoprecipitation experiment. (C) Expected results of co-immunoprecipitation whereas CL16 interferes with interactions between RhoA and ARHGEF1.

Article Snippet: RhoA Pull-down Activation Assay Biochem Kit (bead pull-down format) , Cytoskeleton , Cat# BK036.

Techniques: Inhibition, Activity Assay, Activation Assay, Immunoprecipitation

Journal: Journal of Orthopaedic Translation

Article Title: RhoA deficiency in chondrocyte inhibits cartilage fibrosis and ameliorates osteoarthritis progression via SOX4/MMP2 axis

doi: 10.1016/j.jot.2026.101127

Figure Lengend Snippet: Analysis of potential regulatory molecules involved in cartilage fibrosis based on the single-cell sequencing database of cartilage tissue of OA patients. a, b. Differences in the quantity and distribution of cartilage cell subpopulations between the Control and OA groups. c. GSEA enrichment analysis of significantly altered characteristic KEGG signaling pathways in the fibrocartilage chondrocytes. d. GSEA enrichment analysis of the main molecular alterations in the fibrocartilage chondrocytes cytoskeleton regulatory pathway (hsa04810: Regulation of actin cytoskeleton). e. The AddModuleScore algorithm assigns scores to the KEGG and GO pathways related to the cytoskeleton, and the mapping graph shows the distribution and expression levels of pathway information in each chondrocyte subpopulation. f. Expression and distribution differences of RHOA in each cartilage cell subpopulation between the Control and OA groups, and analysis of RHOA expression in the non-weight-bearing and weight-bearing areas of the femoral medial condyle. g. Bubble chart shows RHOA expression intensity in each cell subpopulation.

Article Snippet: Col2a1 −CreERT and Rhoa -flox/flox strains were obtained from Sangon Biotech (Suzhou, China).

Techniques: Single Cell, Sequencing, Control, Protein-Protein interactions, Expressing

Journal: Journal of Orthopaedic Translation

Article Title: RhoA deficiency in chondrocyte inhibits cartilage fibrosis and ameliorates osteoarthritis progression via SOX4/MMP2 axis

doi: 10.1016/j.jot.2026.101127

Figure Lengend Snippet: RhoA may regulate chondrocyte biological functions, influencing OA progression. a. Imaging of the patient's knee joint to assess OA severity. b, c. Immunofluorescence staining and Western blotting show the RhoA expression in the cartilages of control and OA patient. d, e. RhoA immunofluorescence staining and Western blotting in control and OA mouse cartilage tissue. ∗P < 0.05.

Article Snippet: Col2a1 −CreERT and Rhoa -flox/flox strains were obtained from Sangon Biotech (Suzhou, China).

Techniques: Imaging, Immunofluorescence, Staining, Western Blot, Expressing, Control

Journal: Journal of Orthopaedic Translation

Article Title: RhoA deficiency in chondrocyte inhibits cartilage fibrosis and ameliorates osteoarthritis progression via SOX4/MMP2 axis

doi: 10.1016/j.jot.2026.101127

Figure Lengend Snippet: Construction and identification of chondrocytic Rhoa conditional knockout mice. a. Schematic of the transgenic mouse breeding pattern. b. Southern blots show the genotyping of the transgenic mice. c, d. Western blot and immunofluorescence staining illustrate the RhoA expression in cartilage tissue. ∗P < 0.05.

Article Snippet: Col2a1 −CreERT and Rhoa -flox/flox strains were obtained from Sangon Biotech (Suzhou, China).

Techniques: Knock-Out, Transgenic Assay, Western Blot, Immunofluorescence, Staining, Expressing

Journal: Journal of Orthopaedic Translation

Article Title: RhoA deficiency in chondrocyte inhibits cartilage fibrosis and ameliorates osteoarthritis progression via SOX4/MMP2 axis

doi: 10.1016/j.jot.2026.101127

Figure Lengend Snippet: Loss of RhoA in chondrocytes alleviates cartilage matrix destruction in OA mice. a. Quantitative analysis of osteophyte volume and subchondral bone indicators (Tb.N, Tb.Th, Tb.Sp) through micro-CT three-dimensional reconstruction. b. Safranin O-fast green/Toluidine blue staining to assess cartilage matrix loss. c, d. Immunofluorescence staining and Western blot analysis of Collagen II fluorescence intensity and protein expression in cartilage tissue. ∗P < 0.05.

Article Snippet: Col2a1 −CreERT and Rhoa -flox/flox strains were obtained from Sangon Biotech (Suzhou, China).

Techniques: Micro-CT, Staining, Immunofluorescence, Western Blot, Fluorescence, Expressing

Journal: Journal of Orthopaedic Translation

Article Title: RhoA deficiency in chondrocyte inhibits cartilage fibrosis and ameliorates osteoarthritis progression via SOX4/MMP2 axis

doi: 10.1016/j.jot.2026.101127

Figure Lengend Snippet: RhoA deficiency in chondrocytes significantly inhibits cartilage fibrosis. a. Single-cell sequencing of OA joint cartilage tissues from the Cre and cKO groups for subpopulation classification of chondrocytes. b-c. Perform time-series analysis using the Monocle2 algorithm. d. Use the CytoTRACE algorithm to analyze and display the degree of cell differentiation along each branch path of the proposed time-series. The trend from blue to red indicates that the degree of cell differentiation increases from low to high. e. GO functional analysis of each chondrocyte subpopulation to verify classification accuracy. f. Compare the proportions of each cartilage cell subpopulation in the Cre and cKO groups. g. The changes in cell density of each cartilage cell subpopulation in the branching path as depicted in b h. Sirius Red staining with polarized light microscopy (yellow/orange birefringence indicates type I collagen; green indicates type II collagen) showing reduced yellow/orange signal intensity in the cKO group compared to the Cre group. i , j . Immunofluorescence staining of Collagen I and Fibronectin 1 in cartilage tissue. k . Western blot detection of Collagen I, α-SMA, and TGF-β expression in cartilage tissue. l, m. Immunofluorescence analysis of primary chondrocytes isolated from Cre and cKO OA cartilage. l. Collagen I (green) and phalloidin-labeled F-actin (red). m. Fibronectin 1 (red) and phalloidin-labeled F-actin (green). ∗P < 0.05.

Article Snippet: Col2a1 −CreERT and Rhoa -flox/flox strains were obtained from Sangon Biotech (Suzhou, China).

Techniques: Single Cell, Sequencing, Cell Differentiation, Functional Assay, Staining, Light Microscopy, Immunofluorescence, Western Blot, Expressing, Isolation, Labeling

Journal: Journal of Orthopaedic Translation

Article Title: RhoA deficiency in chondrocyte inhibits cartilage fibrosis and ameliorates osteoarthritis progression via SOX4/MMP2 axis

doi: 10.1016/j.jot.2026.101127

Figure Lengend Snippet: MMP2 mediates the role of chondrocytics RhoA in the cartilage fibrosis and OA progression. a-b. Temporal analysis of single-cell sequencing of Cre and cKO chondrocyte tissues, functionally enriching the gene trends regulating the transition from "immune regulation process" and "homeostasis, proliferation" to the "fibrosis process." c. Volcano plot analysis of single-cell sequencing data from Cre and cKO chondrocyte tissues, showing significant upregulation of Mmp2 expression in the fibrocartilage chondrocytes subpopulation. d, e. Personalized analysis of the single-cell sequencing database from OA patient chondrocyte tissues, indicating that Mmp2 is predominantly located in the OA fibrocartilage chondrocytes group ( d ), and is specifically expressed in OA fibrocartilage chondrocytes compared to the Control group ( e ). f. Differential expression volcano plot from single-cell sequencing showing Mmp2 expression status. g, h. Immunofluorescence staining and Western blot detection of MMP2 expression in chondrocyte tissues. i, j. Western blot and Immunofluorescence staining detection of Collagen I expression in the cKO + AAV2-Vector group and cKO + AAV2- Mmp2 group. k. Safranin O-fast green/Toluidine blue staining to detect cartilage matrix loss. l. Micro-CT quantitative analysis of osteophyte volume. ∗P < 0.05.

Article Snippet: Col2a1 −CreERT and Rhoa -flox/flox strains were obtained from Sangon Biotech (Suzhou, China).

Techniques: Single Cell, Sequencing, Expressing, Control, Quantitative Proteomics, Immunofluorescence, Staining, Western Blot, Plasmid Preparation, Micro-CT

Journal: Journal of Orthopaedic Translation

Article Title: RhoA deficiency in chondrocyte inhibits cartilage fibrosis and ameliorates osteoarthritis progression via SOX4/MMP2 axis

doi: 10.1016/j.jot.2026.101127

Figure Lengend Snippet: The β-Catenin/SOX4 axis modulates the regulating of MMP2 expression by RhoA. a. Sequencing results of cartilage single cells from Cre and cKO mice, showing significant WNT signaling pathway activity in the fibrocartilage chondrocytes population. b, c. Immunofluorescence staining and Western blot analysis to detect β-Catenin expression in the cartilage tissues of Cre and cKO mice. d. Analysis of transcription factors in each cell subgroup of Cre and cKO mouse cartilage sequencing. e. TF-Gene regulatory network analysis of transcription factors and gene regulatory networks in the top 10 subpopulations within the fibrocartilage chondrocytes group. f. Personalized analysis of SOX4 expression in each cartilage cell subgroup using the single-cell sequencing database of OA patient cartilage tissues. g. Analysis of the cellular localization and expression levels of Sox4 in Cre and cKO mouse cartilage single cells. h, i. Immunofluorescence staining and Western blot detection of SOX4 expression in the cartilage tissues of Cre and cKO mice. ∗P < 0.05.

Article Snippet: Col2a1 −CreERT and Rhoa -flox/flox strains were obtained from Sangon Biotech (Suzhou, China).

Techniques: Expressing, Sequencing, Activity Assay, Immunofluorescence, Staining, Western Blot, Single Cell

Journal: Journal of Orthopaedic Translation

Article Title: RhoA deficiency in chondrocyte inhibits cartilage fibrosis and ameliorates osteoarthritis progression via SOX4/MMP2 axis

doi: 10.1016/j.jot.2026.101127

Figure Lengend Snippet: Overexpression of Sox4 significantly reversed the inhibitory effect of Rhoa -cKO on fibrosis and increased cartilage matrix degradation. a. Western blot detection of MMP2, SOX4, and Collagen I protein expression levels in cartilage tissue. b. Immunofluorescence staining to detect changes in Collagen I fluorescence intensity in cartilage tissue. c. Quantitative analysis of osteophyte volume changes using Micro-CT. d, e. Detection of cartilage matrix loss using Safranin O-fast green and Toluidine blue staining. f. Western blot analysis of Collagen I expression in primary chondrocytes treated with control medium, IL-1β, or IL-1β+Si- Sox4 . g, h. Immunofluorescence analysis of primary chondrocytes under indicated treatments: g. Collagen I (green) and phalloidin-labeled F-actin (red). h. Fibronectin 1 (red) and phalloidin-labeled F-actin (green). ∗P < 0.05.

Article Snippet: Col2a1 −CreERT and Rhoa -flox/flox strains were obtained from Sangon Biotech (Suzhou, China).

Techniques: Over Expression, Western Blot, Expressing, Immunofluorescence, Staining, Fluorescence, Micro-CT, Control, Labeling

Journal: bioRxiv

Article Title: Microglial lipid signaling drives glioblastoma invasion and represents a therapeutic vulnerability

doi: 10.64898/2026.04.24.720633

Figure Lengend Snippet: (A) Gene expression of Yap-target genes ( Ankrd1, Ctgf, Cyr61 ) in NSCG (left) and NFpp10 (right) cells upon 24 hours of treatment with BV2 CM, BV2 CM LRA (lipid removal), BV2 CM LRA (lipid removal) LPA 18:1. Relative gene expression to S18. Fold increase. NSCG: n=3, 2way ANOVA, ** p-value<0.0056; *** p-value<0.0003, **** p-value<0.0001; NFpp10: n=3, 2way ANOVA, * p-value<0.0381, ** p-value=0.0012, *** p-value=0.0002, **** p-value<0.0001. (B) Representative immunoblots of Yap nuclear and cytoplasmic levels in NFpp10 cells upon 5 minutes, 10 minutes, 20 minutes of co-culture with BV2 cells and LPA 16:0 treatment. (C) Representative immunoblots of RhoA pulldown and total in NSCG (left) and NFpp10 (right) cells upon 2 minutes, 5 minutes, 10 minutes co-culture with BV2 cells. (D) Representative immunoblots of RhoA pulldown and total in NSCG (left) and NFpp10 (right) cells upon 5 minutes, 10 minutes co-culture with BV2 cells and LPA 18:1 or LPA 16:0 treatment respectively. (E) Invasion assays of NSCG (left) and NFpp10 (right) Scr (control) and YAP/TAZ knock-out cells in the presence or absence of BV2 cells. Fold increase. NSCG: n=3, One-way ANOVA, * p-value=0.0267, *** p-value=0.0001; NFpp10: n=6, One-way ANOVA, * p-value=0.0432, ** p-value=0.0034. (F) Invasion assays of NSCG Scr (control) and YAP knock-out cells in the presence or absence of BV2 cells and LPA 18:1 treatment. Fold increase. NSCG: n=3, One-way ANOVA, ** p-value<0.0022, *** p-value=0.0004, **** p-value<0.0001. (G) Tumor cell number across the 50 distinct TMAs. h. Ratio tumor cell/microglia-macrophages across the selected 15 tumor core and 15 tumor rim TMAs based on tumor cell density. i. Correlation tumor cells/microglia-macrophages across the selected 15 tumor core and 15 tumor rim TMAs. Each dot/number refers to a TMA sample. Correlation index: −0.477. All data represent mean ± SEM.

Article Snippet: Protein extraction was performed using the RhoA Pulldown activation Assay Kit (Cytoskeleton, BK036).

Techniques: Gene Expression, Western Blot, Co-Culture Assay, Control, Knock-Out