rhoa inhibitor i  (Cytoskeleton Inc)

Journal: bioRxiv

Article Title: Pre-myelinating oligodendrocyte ADGRG1 is required for axon ensheathment and CNS myelin formation

doi: 10.64898/2026.01.26.701898

Figure Lengend Snippet: (A) Schematic of the proposed signaling pathway showing ADGRG1 activating the RhoA pathway to promote F-actin polymerization in preOLs. (B) Immunostaining of control and cKO preOLs for ADGRG1 (H11, green), GTP-RhoA (magenta), DAPI (blue) and brightfield. Colocalization between ADGRG1 and GTP-RhoA was observed in the processes of control preOLs, but both signals were downregulated in cKO preOLs. Scale bar, 10 μm. (C) Representative images of control and cKO oligodendrocyte cultures differentiated for 3, 5, or 7 days, treated with either vehicle, Rho inhibitor (for control), or Rho activator (for cKO). Cultures were stained for MBP (magenta), BCAS1 (green), Olig2 (gray), and DAPI (blue). Scale bar, 10 μm. (D) Quantification of the percentage of BCAS1 + Olig2 + cells in total Olig2 + cells across differentiation time points. Each circle represents one biological replicate. ** p=0.0011 (diff 3 day Con vs cKO), ** p=0.0011 (diff 3 day cKO vs cKO + Rho activator), ** p=0.0088 (diff 5 day Con vs Con + Rho inhibitor), * p=0.0103 (diff 5 day Con vs cKO), * p=0.0181 (diff 5 day cKO vs cKO + Rho activator), *** p=0.0005 (diff 7 day Con vs cKO), *** p=0.0003 (diff 7 day cKO vs cKO + Rho activator). Two-way ANOVA with Bonferroni’s multiple comparisons test. (E) Quantification of the percentage of MBP + Olig2 + cells in total Olig2 + cells across differentiation time points. Each circle represents one biological replicate. **** p<0.0001 (diff 5 day Con vs Con + Rho inhibitor), ** p=0.0047 (diff 5 day Con vs cKO), * p=0.0108 (diff 5 day cKO vs cKO + Rho activator), **** p<0.0001 (diff 7 day Con vs Con + Rho inhibitor), ** p=0.0010 (diff 7 day Con vs cKO), **** p<0.0001 (diff 7 day cKO vs cKO + Rho activator). Two-way ANOVA with Bonferroni’s multiple comparisons test. Data are presented as means ± SEM.

Article Snippet: The following compounds were used: RhoA Activator II (1 μg/mL, Cytoskeleton Inc., Cat. #: CN03-A) and RhoA Inhibitor I (1 μg/mL, Cytoskeleton Inc., Cat. #: CT04-A).

Techniques: Immunostaining, Control, Staining

Journal: Journal of Orthopaedic Translation

Article Title: NLRP3 inflammasome impairs fracture repair in Rheumatoid arthritis through RhoA/Rac1-IL1β axis-mediated suppression of osteoblast differentiation

doi: 10.1016/j.jot.2025.07.012

Figure Lengend Snippet: NLRP3 inflammasome mediates osteoblast differentiation in MPCs through the RhoA/Rac1‒IL-1β pathway . (A) WT and NLRP3 KO MPCs were stimulated with or without 20 ng/mL TNFα, cultured in osteoblast-inducing medium for 7 days, and then subjected to ALP staining, and ALP-positive areas (%) were measured. (B) Similarly, WT MPCs were stimulated with or without 20 ng/mL TNFα ± 0.1 μmol/L MCC950 under the same conditions, followed by ALP staining and quantification. (C–D) WT MPCs were stimulated with or without 20 ng/mL TNFα ± MCC950. Representative IF staining of MPCs labeled with anti-NLRP3, anti-IL-1β (C), anti-Runx2 and anti-OCN (D) was shown, with data for integrated optical density (IOD) respectively. (E) The expression of Rac1/ERK signaling molecules, RhoA/ROCK signaling molecules, NLRP3 signaling molecules, Osteoblastogenesis-related molecules, and NF-κB signaling molecules were determined by Western blot analysis in cell lysates of MPCs. (F) WT MPCs were stimulated with or without TNFα, in the presence or absence of 1.0 μg/ml C3 and 50 μM NSC 23766, and the expression levels of the aforementioned signaling molecules were analyzed by Western blot. All the representative immunoblots were quantified by densitometric analysis. Each experiment was performed three to five times. Representative images and quantifications shown in the figure came from one independent experiment. One-way ANOVA followed by Dunnett's post-hoc multiple comparisons were performed. Data are represented as means ± SDs, N = 5, ∗ p < 0.05 vs. Veh, # p < 0.05 vs. TNFα -treatment groups.

Article Snippet: Where indicated, cells were pretreated with the NLRP3 inhibitor MCC950 (MCE, Cat#HY-12815), RhoA inhibitor C3 transferase (Cytoskeleton, Cat#CT-04), Rac1 inhibitor NSC 23766 trihydrochloride (Selleck, Cat#S8031), or ATP (Sigma–Aldrich, Cat#A6419).

Techniques: Cell Culture, Staining, Labeling, Expressing, Western Blot

Journal: Journal of Orthopaedic Translation

Article Title: NLRP3 inflammasome impairs fracture repair in Rheumatoid arthritis through RhoA/Rac1-IL1β axis-mediated suppression of osteoblast differentiation

doi: 10.1016/j.jot.2025.07.012

Figure Lengend Snippet: Schematic representation of the NLRP3 inflammasome delaying fracture healing in RA mice through RhoA/Rac1‒IL-1β-mediated osteoblast differentiation . During fracture healing process in RA mice, NLRP3 in MPCs is activated and initiates the assembly of an inflammasome, through RhoA/Rac1 and NF-κB signaling pathway, resulting in the release of IL-1β. Next, the increased expression of IL-1β inhibits osteoblasts differentiation, leading to delayed fracture healing. Importantly, MCC950 promotes fracture healing by inhibiting NLRP3 inflammasome activation in RA mice.

Article Snippet: Where indicated, cells were pretreated with the NLRP3 inhibitor MCC950 (MCE, Cat#HY-12815), RhoA inhibitor C3 transferase (Cytoskeleton, Cat#CT-04), Rac1 inhibitor NSC 23766 trihydrochloride (Selleck, Cat#S8031), or ATP (Sigma–Aldrich, Cat#A6419).

Techniques: Expressing, Activation Assay

Journal: Scientific Reports

Article Title: Antisense mediated blockade of Dickkopf 1 attenuates tumor survival, metastases and bone damage in experimental osteosarcoma

doi: 10.1038/s41598-024-84037-4

Figure Lengend Snippet: Transcriptional blockade of Dkk-1 in MG63 OS cells attenuates proliferation, survival, osteoinhibition and ALDH1A1 expression. ( A ) ELISA measurement of Dkk-1 in media supernatants by MG63-kdDkk1 and MG63-scr cells. ( B ) Relative transcription of Dkk-1 by MG63-kdDkk1 and MG63-scr cells. ( C ) Cell density at the time of assay for panel A and B. ( D ) Proliferation under standard culture conditions. ( E ) Final cell density of MG63-kdDkk1 and MG63-scr cells after 10 days of nutrient (medium) deprivation under confluent conditions. ( F ) Final cell density of MG63-kdDkk1 and MG63-scr cells after 2 days of exposure to DRB. ( G ) Immunoblots of the phosphorylation status of RhoA, Mkk4, p30 and JNK in MG63-kdDkk1 and MG63-scr cells after 8 days of standard culture. ( H ) Immunoblot assays of ALDH1A1 from triplicate cultures of MG63-kdDkk1 and MG63-scr cells after 8 days of standard culture (above). Densitometric measurements of the triplicate bands normalized to GAPDH (below). ( I ) ELISA measurement of OPG in media supernatants by MG63-kdDkk1 and MG63-scr cells exposed to 5 days of osteogenic conditions. ( J ) Colorimetric assay of ALP activity in MG63-kdDkk1 and MG63-scr monolayers exposed to 8 days of osteogenic conditions. ( K ) ARS-stained monolayers of MG63-kdDkk1 and MG63-scr monolayers exposed to 5, 10 and 15 days of mineralizing osteogenic conditions. ( L ) Spectrophotometric measurement of ARS stain recovered from monolayers in Panel K. Statistics: Panel A-C, E, H-J: two-tailed student’s T - test. **p < 0.01, ***p < 0.005, n = as indicated. Panel D, L: two-sided ANOVA. Panel F: one-sided ANOVA and Tukey’s multiple comparison test. ***p < 0.005, n = 6.

Article Snippet: CT04 cell permeable RhoA inhibitor was acquired from Cytoskeleton Inc. (Denver, CO).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Phospho-proteomics, Colorimetric Assay, Activity Assay, Staining, Two Tailed Test, Comparison

Journal: Scientific Reports

Article Title: Antisense mediated blockade of Dickkopf 1 attenuates tumor survival, metastases and bone damage in experimental osteosarcoma

doi: 10.1038/s41598-024-84037-4

Figure Lengend Snippet: List of agents used in MOSJ-Dkk1 assays.

Article Snippet: CT04 cell permeable RhoA inhibitor was acquired from Cytoskeleton Inc. (Denver, CO).

Techniques: Phospho-proteomics, Activity Assay, Binding Assay, Activation Assay

Journal: Scientific Reports

Article Title: Antisense mediated blockade of Dickkopf 1 attenuates tumor survival, metastases and bone damage in experimental osteosarcoma

doi: 10.1038/s41598-024-84037-4

Figure Lengend Snippet: Summarized outcome of MOSJ-Dkk1 assays.

Article Snippet: CT04 cell permeable RhoA inhibitor was acquired from Cytoskeleton Inc. (Denver, CO).

Techniques:

Journal: Nature Communications

Article Title: Soluble TREM2 ameliorates tau phosphorylation and cognitive deficits through activating transgelin-2 in Alzheimer’s disease

doi: 10.1038/s41467-023-42505-x

Figure Lengend Snippet: a Schematic illustration of the potential mechanism by which sTREM2 regulates tau phosphorylation: sTREM2 secreted from microglia binds neuronal TG2 and inactivates the RhoA/ROCK/GSK3β pathway, attenuating tau hyperphosphorylation. b , c Western blots showing the phosphorylation of tau and GSK3β in HEK293-Tau cells treated with control (Fc, 20 nM), Fc-sTREM2 (20 nM), or Tat-C3 (RhoA inhibitor, 0.1 μg/mL) for 24 h. d , e Western blots showing the phosphorylation of tau and GSK3β in HEK293-Tau cells treated with control (Fc, 20 nM), Fc-sTREM2 (20 nM), or Y-27632 (ROCK inhibitor) for 24 h. f , g Western blots showing the phosphorylation of tau and GSK3β in HEK293-Tau cells treated with control (Fc, 20 nM), Fc-sTREM2 (20 nM) for 24 h and incubated with RhoA activator (1 U/ml) or solvent for another 30 min. EK293-Tau cells were pretreated with control siRNA (si-NC), si-RhoA ( h , i ), or si-ROCK ( j , k ), and then treated with control (Fc, 20 nM) or Fc-sTREM2 (20 nM). Shown are the phosphorylation of tau and GSK3β (mean ± s.e.m.; one-way ANOVA for ( c , e , g ), two-way ANOVA for ( i , k ), n = 4 independent experiments; compared with the control group, * P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: To test the role of the Rho-ROCK pathway in tau phosphorylation, HEK293-tau cells were treated with the control (Fc, 20 nM), sTREM2 (20 nM), RhoA inhibitor Tat-C3 (0.1 μg/ml, Cytoskeleton, CT03) or the ROCK inhibitor Y-27632 (25 nM, Santa Cruz, sc-3536) for 24 h. RhoA activator (1 U/ml, Cytoskeleton, CN01) was added to HEK293-tau cells after the cells were treated with Fc-sTREM2 (20 nM) for 24 h and incubated for another 30 min before analysis.

Techniques: Phospho-proteomics, Western Blot, Control, Incubation, Solvent