Journal: Journal of Cell Science

Article Title: Prostaglandin E2 inhibits adipogenesis through the cilia-dependent activation of ROCK2

doi: 10.1242/jcs.264193

Figure Lengend Snippet: Ciliary EP4 does not signal via ciliary cAMP. (A) 3T3-L1 primary cilia lengths measured at 0-, 24- and 48-h time points of adipogenesis with or without 20 µM PGE2. Unfilled circles denote lengths of individual cilia measured across four independent trials; filled circles represent the average ciliary length per trial. Mean of all four averages±s.d. shown. n =total number of cilia measured per condition across all four trials. (B) Undifferentiated 3T3-L1 cells expressing cilia-targeted cAMP biosensor treated with DMSO (black), the adenylyl cyclase activator forskolin (50 µM, blue), the FFAR4 agonist TUG891 (50 µM, green) or PGE2 (20 µM, red) at the time point indicated by the arrow; images collected every 20 s were used to calculate the ratio of fluorescence intensities between the constitutive ciliary marker and the cAMP sensor at each time point. Data are average of three independent trials ±s.e.m. n =total number of experimental wells; N =total number of cilia analyzed for each condition. (B′) Representative images of offset cAMP sensor (green) and constitutive ciliary marker (red) at the indicated time points. (C) 3T3-L1 cells treated with different PKA inhibitors (25 µM Rp-cAMPs, 25 µM Rp-8-Br-cAMPs and 10 µM H89) in the presence or absence of PGE2 during the first 96 h of adipogenesis. Dashed line marks the average endpoint lipid content in vehicle-treated control cells treated with PGE2. Only H89 rescues adipogenesis in the presence of PGE2. (D) Heat map of percentage activity remaining for different kinase targets in response to indicated kinase inhibitors. Green text denotes inhibitors that rescue adipogenesis in the presence of PGE2 (see Fig. S4E ). (E) 3T3-L1 cells differentiated in the presence or absence of 20 µM PGE2 and the kinase inhibitors H89 (10 µM) or GSK429286A (1 µM) during the first 48 h of adipogenesis. Both inhibitors rescue adipogenesis in the presence of PGE2 and inhibit ROCK2 to similar extents. (C,E) Data are mean±s.d. and each data point represents an independent experiment. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001 (one-way ANOVA followed by Tukey's multiple comparison test). a.u., arbitrary units; ns, not significant.

Article Snippet: TaqMan probes targeting Pparg (Thermo Fisher Scientific, MM01184322_M1), Cebpa (Thermo Fisher Scientific, Mm00514283_s1), Adipoq (Thermo Fisher Scientific, Mm04933656_m1), Fabp4 (Thermo Fisher Scientific, Mm00445878_m1), Rock1 (Thermo Fisher Scientific, Mm00485733_m1), Rock2 (Thermo Fisher Scientific, Mm01270843_m1) and the housekeeping gene Nono (Thermo Fisher Scientific, MM00834875_G1) were amplified using TaqMan Gene expression master mix (Thermo Fisher Scientific, 4369016) in MicroAmp Optical 384-well reaction plates (Applied Biosystems, 4309849) according to manufacturer protocols.

Techniques: Expressing, Fluorescence, Marker, Control, Activity Assay, Comparison

Journal: Journal of Cell Science

Article Title: Prostaglandin E2 inhibits adipogenesis through the cilia-dependent activation of ROCK2

doi: 10.1242/jcs.264193

Figure Lengend Snippet: PGE2 activates ROCK2 in a cilia-dependent manner to inhibit adipogenesis. (A) 3T3-L1 cells treated with 0.5 µg/ml Rho Inhibitor I during the first 48 h partially rescues PGE2 co-treatment. Left: Endpoint lipid content with dashed line marking average lipid content of vehicle-treated control cells treated with PGE2. Right: Representative images at endpoint of adipogenesis with lipid droplets stained with BODIPY. (B) 3T3-L1 cells treated with 0.25 µg/ml Rho Activator II during the first 48 h of adipogenesis inhibits differentiation with similar efficacy as PGE2. Left: Endpoint lipid content for each condition. Right: Representative images at endpoint of adipogenesis with lipid droplets stained with BODIPY. (C) Actin network of 3T3-L1 cells visualized by phalloidin staining (red). Undifferentiated 3T3-L1 cells predominantly have actin stress fibers, which are disassembled within 48 h of adipogenesis initiation. Stress fibers are not disassembled in the presence of 20 µM PGE2, and stress fiber disassembly is rescued with ROCK2 inhibitor co-treatment (1 µM GSK429286A). Representative images of each treatment condition. Boxed areas are shown at higher magnification below. (D) Violin plot quantifying phalloidin staining intensity in 3T3-L1 cells. (E) Actin networks of TULP3 knockout cells visualized by phalloidin staining. TULP3 knockouts lacking ciliary EP4 disassemble actin stress fibers during adipogenesis regardless of PGE2 treatment or ROCK inhibition. Representative images of each treatment condition. (F) Quantification of phalloidin staining intensity in TULP3 knockout cells. (G) 20 µM PGE2 treatment does not prevent actin stress fiber disassembly in EP4 knockout cells. Representative images of each treatment condition. (H) Quantification of phalloidin staining intensity in EP4 knockout cells. (I) Model for how PGE2 inhibits adipogenesis. In wild-type 3T3-L1 cells and ASCs, activation of ciliary EP4 by PGE2 activates RhoA/ROCK2, which stabilizes the actin cytoskeleton and prevents adipogenesis. Ciliary localization of EP4 is required for this activity. Created in BioRender by Lee, M., 2025. https://BioRender.com/t962fv5 . This figure was sublicensed under CC-BY 4.0 terms. (C,E,G) Boxed areas are shown at higher magnification below. (D,F,H) Violin plots quantify phalloidin staining intensity from 18 images per condition collected equally across three independent repeats. Solid lines within each violin depict the median, while dotted lines are quartiles. Dashed lines across the graph indicate the average phalloidin staining in vehicle treated control cells after 48 h of adipogenesis. (A,B) Data are mean±s.d. and each data point represents an independent experiment. (A,B,D,F,H) * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001 (one-way ANOVA followed by Tukey's multiple comparison test). a.u., arbitrary units; ns, not significant.

Article Snippet: TaqMan probes targeting Pparg (Thermo Fisher Scientific, MM01184322_M1), Cebpa (Thermo Fisher Scientific, Mm00514283_s1), Adipoq (Thermo Fisher Scientific, Mm04933656_m1), Fabp4 (Thermo Fisher Scientific, Mm00445878_m1), Rock1 (Thermo Fisher Scientific, Mm00485733_m1), Rock2 (Thermo Fisher Scientific, Mm01270843_m1) and the housekeeping gene Nono (Thermo Fisher Scientific, MM00834875_G1) were amplified using TaqMan Gene expression master mix (Thermo Fisher Scientific, 4369016) in MicroAmp Optical 384-well reaction plates (Applied Biosystems, 4309849) according to manufacturer protocols.

Techniques: Control, Staining, Knock-Out, Inhibition, Activation Assay, Activity Assay, Comparison

Journal: Journal of Translational Medicine

Article Title: Triad of homocysteine, ROCK2 and vimentin as a convergent biomarker signature in pseudoexfoliation

doi: 10.1186/s12967-025-07437-8

Figure Lengend Snippet: Plasma levels of homocysteine, ROCK2, and vimentin in pseudoexfoliation syndrome and glaucoma. Column scatter plots display quantified plasma concentrations of ( A ) Hcy, ( B ) ROCK2, and ( C ) VIM which were significantly higher in the PEX (PEXS + PEXG) group ( n = 48), as well as in both PEXS and PEXG subgroups ( n = 24 each), compared to controls ( n = 24). Kruskal–Wallis tests revealed significant group-wise differences for all markers ( p < 0.001 for Hcy and VIM, p = 0.001 for ROCK2). Dunn’s post hoc test confirmed elevated levels in PEXS and PEXG vs. controls ( p < 0.01), with no significant differences between the two PEX subgroups. Data are presented as mean ± SD

Article Snippet: Plasma levels of VIM and ROCK2 were measured using commercial ELISA kits (Human Vimentin ELISA Kit, CSB-E08982h; and Human ROCK2 ELISA Kit, CSB-E14993h; both from CUSABIO) according to the manufacturer’s protocols.

Techniques: Clinical Proteomics

Journal: Journal of Translational Medicine

Article Title: Triad of homocysteine, ROCK2 and vimentin as a convergent biomarker signature in pseudoexfoliation

doi: 10.1186/s12967-025-07437-8

Figure Lengend Snippet: Distribution analysis and effect sizes of plasma biomarkers across study groups. ( A–C ) Q–Q plots display the distribution of plasma Hcy, ROCK2, and VIM levels in Control, PEXS, and PEXG groups relative to a theoretical normal distribution. Deviations from the reference line (y = x) indicate non-normality. ( D ) Violin plots show biomarker distribution and effect sizes across group comparisons: Control vs. PEX group, Control vs. PEXS, Control vs. PEXG, and PEXS vs. PEXG. Boxplots indicate medians and interquartile ranges. Cohen’s d values quantify effect sizes. Hcy and VIM showed large effects and consistent upregulation across disease groups, while ROCK2 showed moderate elevation. Minimal separation was observed between PEXS and PEXG for all markers

Article Snippet: Plasma levels of VIM and ROCK2 were measured using commercial ELISA kits (Human Vimentin ELISA Kit, CSB-E08982h; and Human ROCK2 ELISA Kit, CSB-E14993h; both from CUSABIO) according to the manufacturer’s protocols.

Techniques: Clinical Proteomics, Control, Biomarker Discovery

Journal: Journal of Translational Medicine

Article Title: Triad of homocysteine, ROCK2 and vimentin as a convergent biomarker signature in pseudoexfoliation

doi: 10.1186/s12967-025-07437-8

Figure Lengend Snippet: Receiver operating characteristic curves (ROC) of plasma homocysteine, ROCK2, and Vimentin for distinguishing PEXS and PEXG from control subjects. ( A - C ) Hcy showed excellent diagnostic performance with AUCs of 0.98 (95% CI: 0.95–1.00) for Control vs. PEX (PEXS + PEXG), 0.98 (95% CI: 0.95–1.00) for Control vs. PEXS, and 0.97 (95% CI: 0.92–1.00) for Control vs. PEXG. Optimal cut-off values were > 15.09 µmol/L, > 15.19 µmol/L, and > 21.40 µmol/L, respectively. (D-F) ROC analysis of ROCK2 yielded AUCs of 0.77 (95% CI: 0.66–0.88), 0.74 (95% CI: 0.60–0.88), and 0.80 (95% CI: 0.68–0.93) for Control vs. PEX (PEXS + PEXG), PEXS, and PEXG, with corresponding cut-offs of > 375.1 pg/mL, > 378.5 pg/mL, and > 353.1 pg/mL. (G-I) VIM showed AUCs of 0.94 (95% CI: 0.88–0.99), 0.89 (95% CI: 0.79–0.99), and 0.99 (95% CI: 0.97–1.00) for Control vs. PEX (PEXS + PEXG), PEXS, and PEXG, with optimal cut-offs of > 484.60 pg/mL, > 467.30 pg/mL, and > 486.30 pg/mL, respectively

Article Snippet: Plasma levels of VIM and ROCK2 were measured using commercial ELISA kits (Human Vimentin ELISA Kit, CSB-E08982h; and Human ROCK2 ELISA Kit, CSB-E14993h; both from CUSABIO) according to the manufacturer’s protocols.

Techniques: Clinical Proteomics, Control, Diagnostic Assay

Journal: Journal of Translational Medicine

Article Title: Triad of homocysteine, ROCK2 and vimentin as a convergent biomarker signature in pseudoexfoliation

doi: 10.1186/s12967-025-07437-8

Figure Lengend Snippet: Correlation analyses between homocysteine, ROCK2, and Vimentin. ( A ) Global correlation matrix for all the study subjects shows strong positive correlation between expression of Hcy and ROCK2 (ρ = 0.62), ROCK2 and VIM (ρ = 0.69), VIM and Hcy (ρ = 0.79). ( B ) Stratified correlation analysis showing preservation of associations within individual groups with varying strength. ( C ) Dot plot displaying Spearman correlation coefficients (ρ) for each biomarker pair: ROCK2 and VIM, ROCK2 and Hcy, and VIM and Hcy across the three study groups. Strong positive correlations were observed among all pairs in PEXS and PEXG, while correlations in the control group were weak or absent. Notably, the correlation between ROCK2 and VIM increases in PEXS but declines in PEXG. In contrast, the strength of association between Hcy and both ROCK2 and VIM increases progressively from PEXS to PEXG. The dashed line at ρ = 0 indicates no correlation

Article Snippet: Plasma levels of VIM and ROCK2 were measured using commercial ELISA kits (Human Vimentin ELISA Kit, CSB-E08982h; and Human ROCK2 ELISA Kit, CSB-E14993h; both from CUSABIO) according to the manufacturer’s protocols.

Techniques: Expressing, Preserving, Biomarker Discovery, Control

Journal: Journal of Translational Medicine

Article Title: Triad of homocysteine, ROCK2 and vimentin as a convergent biomarker signature in pseudoexfoliation

doi: 10.1186/s12967-025-07437-8

Figure Lengend Snippet: Effect of elevated homocysteine on ROCK2 and VIM in HLE-B3 cells. ( A - B ) Representative immunoblots showing ROCK2 and VIM protein levels in HLE-B3 cells treated with 0 (Vehicle Control), 500 µM, or 1000 µM Hcy for 48 h. GAPDH was used as a loading control. ( C – D ) Quantitative densitometric analysis of ROCK2 ( C ) and VIM ( D ) normalized to GAPDH. Data are presented as mean ± SD from five independent biological replicates ( n = 5). Treatment with Hcy led to a concentration-dependent increase in expression of both markers. Statistical analysis was performed using the Mann–Whitney U test. Significance is indicated as follows: p < 0.05 (*), and p < 0.01 (**)

Article Snippet: Plasma levels of VIM and ROCK2 were measured using commercial ELISA kits (Human Vimentin ELISA Kit, CSB-E08982h; and Human ROCK2 ELISA Kit, CSB-E14993h; both from CUSABIO) according to the manufacturer’s protocols.

Techniques: Western Blot, Control, Concentration Assay, Expressing, MANN-WHITNEY

Journal: Journal of Translational Medicine

Article Title: Triad of homocysteine, ROCK2 and vimentin as a convergent biomarker signature in pseudoexfoliation

doi: 10.1186/s12967-025-07437-8

Figure Lengend Snippet: Diagnostic performance of two-marker and three-marker biomarker models. Receiver operating characteristic (ROC) curves comparing the classification performance of different biomarker combinations for diagnostic accuracy. ( A ) Two-marker model with Hcy and VIM achieved an AUC of 0.98 (95% CI: 0.95–1.00), a cut-off of 0.80, sensitivity of 0.91, and specificity of 0.83. ( B ) Two-marker model with Hcy and ROCK2 showed an AUC of 0.97 (95% CI: 0.94–1.00), with a cut-off of 0.48, sensitivity of 0.94, and specificity of 0.83. ( C ) Two-marker model with VIM and ROCK2 resulted in an AUC of 0.94 (95% CI: 0.88–0.99), a cut-off of 0.47, sensitivity of 0.88, and specificity of 0.80. ( D ) Three-marker model combining Hcy, ROCK2, and VIM yielded comparable diagnostic performance, with an AUC of 0.98 (95% CI: 0.96–1.00), but with a balanced cut-off score of 0.77, sensitivity of 0.94, and specificity of 0.88

Article Snippet: Plasma levels of VIM and ROCK2 were measured using commercial ELISA kits (Human Vimentin ELISA Kit, CSB-E08982h; and Human ROCK2 ELISA Kit, CSB-E14993h; both from CUSABIO) according to the manufacturer’s protocols.

Techniques: Diagnostic Assay, Marker, Biomarker Discovery

Journal: Journal of Translational Medicine

Article Title: Triad of homocysteine, ROCK2 and vimentin as a convergent biomarker signature in pseudoexfoliation

doi: 10.1186/s12967-025-07437-8

Figure Lengend Snippet: Receiver Operating Characteristic (ROC) curves for individual and combined biomarker models distinguishing study groups. ( A ) Control vs. PEX (PEXS + PEXG), ( B ) Control vs. PEXS, ( C ) Control vs. PEXG, ( D ) PEXS vs. PEXG. Each panel shows ROC curves for models based on individual biomarkers: Homocysteine (blue), ROCK2 (orange), Vimentin (green), and a combined LASSO logistic regression model (red). LASSO models were trained using repeated 5-fold cross-validation, and optimism-corrected AUCs were estimated using 1000 bootstrap replicates. AUC values are displayed within each panel

Article Snippet: Plasma levels of VIM and ROCK2 were measured using commercial ELISA kits (Human Vimentin ELISA Kit, CSB-E08982h; and Human ROCK2 ELISA Kit, CSB-E14993h; both from CUSABIO) according to the manufacturer’s protocols.

Techniques: Biomarker Discovery, Control

Journal: Journal of Translational Medicine

Article Title: Triad of homocysteine, ROCK2 and vimentin as a convergent biomarker signature in pseudoexfoliation

doi: 10.1186/s12967-025-07437-8

Figure Lengend Snippet: Calibration and clinical utility of the combined biomarker prediction model. ( A ) Calibration plot for the logistic regression model combining Hcy, ROCK2, and VIM. The x-axis shows predicted disease probability, and the y-axis indicates observed frequency. The navy-blue curve represents the bias-corrected calibration (1000 bootstrap resamples); the diagonal grey dashed line indicates perfect prediction. The model demonstrated good calibration, with a mean absolute error of 0.04 and a mean squared error of 0.003. ( B ) Decision Curve Analysis (DCA) assessing the net clinical benefit of the combined biomarker model. The standardized net benefit is plotted against a range of threshold probabilities. The blue line denotes the biomarker model; the red dashed and gray lines represent the “Treat All” and “Treat None” strategies, respectively. The model shows superior net benefit across the clinically relevant threshold range (0.1–0.8), supporting its potential for individualized, risk-based decision-making

Article Snippet: Plasma levels of VIM and ROCK2 were measured using commercial ELISA kits (Human Vimentin ELISA Kit, CSB-E08982h; and Human ROCK2 ELISA Kit, CSB-E14993h; both from CUSABIO) according to the manufacturer’s protocols.

Techniques: Biomarker Discovery

Journal: Journal of Translational Medicine

Article Title: Triad of homocysteine, ROCK2 and vimentin as a convergent biomarker signature in pseudoexfoliation

doi: 10.1186/s12967-025-07437-8

Figure Lengend Snippet: k-Means (k = 3) clustering of subjects based on biomarker profiles and mean biomarker expression based on z-score. ( A ) PCA plot showing three clusters derived from k-means clustering of standardized Hcy, ROCK2, and VIM values. Clusters show differential enrichment of disease states, with Cluster 3 predominantly composed of controls, Cluster 1 enriched in PEXS, and Cluster 2 enriched in PEXG. Subjects are coloured by disease status and shaped by cluster label. The ellipses represent the 95% confidence boundaries for each cluster. ( B ) Mean biomarker expression (z-score ± SEM) across three unsupervised clusters. Cluster 2 shows coordinated upregulation of Hcy, ROCK2, and VIM, consistent with advanced disease (PEXG). Cluster 3 exhibits downregulated expression typical of controls. Cluster 1 shows mild elevations, suggesting early disease activity (PEXS)

Article Snippet: Plasma levels of VIM and ROCK2 were measured using commercial ELISA kits (Human Vimentin ELISA Kit, CSB-E08982h; and Human ROCK2 ELISA Kit, CSB-E14993h; both from CUSABIO) according to the manufacturer’s protocols.

Techniques: Biomarker Discovery, Expressing, Derivative Assay, Activity Assay

Journal: Journal of Translational Medicine

Article Title: Triad of homocysteine, ROCK2 and vimentin as a convergent biomarker signature in pseudoexfoliation

doi: 10.1186/s12967-025-07437-8

Figure Lengend Snippet: Integrated pathway enrichment analysis of homocysteine, VIM, and ROCK2. ( A ) Bubble plot showing significantly enriched pathways based on –log₁₀(p-value) and pathway impact. Bubble size reflects the number of matched features; colour indicates functional categories. Key pathways highlight redox imbalance, cytoskeletal disruption, and vascular dysfunction. ( B ) Clustered bar plot grouping enriched pathways ( p < 0.05) into functional categories. The most enriched categories were methylation/redox metabolism, cytoskeletal remodelling, and immune signaling, suggesting a central role for metabolic and mechanical stress in PEX pathogenesis

Article Snippet: Plasma levels of VIM and ROCK2 were measured using commercial ELISA kits (Human Vimentin ELISA Kit, CSB-E08982h; and Human ROCK2 ELISA Kit, CSB-E14993h; both from CUSABIO) according to the manufacturer’s protocols.

Techniques: Functional Assay, Disruption, Methylation

Journal: Journal of Translational Medicine

Article Title: Triad of homocysteine, ROCK2 and vimentin as a convergent biomarker signature in pseudoexfoliation

doi: 10.1186/s12967-025-07437-8

Figure Lengend Snippet: STITCH network of PEX-associated genes and homocysteine metabolism. The network includes VIM, ROCK2, their interactors, and homocysteine-metabolizing enzymes. Nodes represent proteins/metabolites; edges indicate functional associations. Distinct modules include cytoskeletal/adhesion (VIM, ROCK1/2, RHOA), methylation/redox (MTHFR, CBS, DNMTs), TGF-β/WNT signaling (TGFB1, ZEB1/2, CTNNB1), and stress response (CASP3, HSPA1A, UBB), illustrating converging pathways in PEX pathogenesis

Article Snippet: Plasma levels of VIM and ROCK2 were measured using commercial ELISA kits (Human Vimentin ELISA Kit, CSB-E08982h; and Human ROCK2 ELISA Kit, CSB-E14993h; both from CUSABIO) according to the manufacturer’s protocols.

Techniques: Functional Assay, Methylation

Journal: Journal of Translational Medicine

Article Title: Triad of homocysteine, ROCK2 and vimentin as a convergent biomarker signature in pseudoexfoliation

doi: 10.1186/s12967-025-07437-8

Figure Lengend Snippet: SP1 as a key transcriptional regulator in Pseudoexfoliation. ( A ) Transcription factor enrichment using TRRUST and ChEA3 identified SP1 as a top-scoring regulator of PEX-associated genes ( B–C ). In silico prediction of putative SP1 binding sites performed within the proximal promoter regions (− 1500 bp upstream of the TSS) of VIM and ROCK2 using the UCSC Genome Browser and JASPAR (motif MA0079.3)

Article Snippet: Plasma levels of VIM and ROCK2 were measured using commercial ELISA kits (Human Vimentin ELISA Kit, CSB-E08982h; and Human ROCK2 ELISA Kit, CSB-E14993h; both from CUSABIO) according to the manufacturer’s protocols.

Techniques: In Silico, Binding Assay

Journal: Journal of Translational Medicine

Article Title: Triad of homocysteine, ROCK2 and vimentin as a convergent biomarker signature in pseudoexfoliation

doi: 10.1186/s12967-025-07437-8

Figure Lengend Snippet: Plasma levels of homocysteine, ROCK2, and vimentin in pseudoexfoliation syndrome and glaucoma. Column scatter plots display quantified plasma concentrations of ( A ) Hcy, ( B ) ROCK2, and ( C ) VIM which were significantly higher in the PEX (PEXS + PEXG) group ( n = 48), as well as in both PEXS and PEXG subgroups ( n = 24 each), compared to controls ( n = 24). Kruskal–Wallis tests revealed significant group-wise differences for all markers ( p < 0.001 for Hcy and VIM, p = 0.001 for ROCK2). Dunn’s post hoc test confirmed elevated levels in PEXS and PEXG vs. controls ( p < 0.01), with no significant differences between the two PEX subgroups. Data are presented as mean ± SD

Article Snippet: Plasma levels of VIM and ROCK2 were measured using commercial ELISA kits (Human Vimentin ELISA Kit, CSB-E08982h; and Human ROCK2 ELISA Kit, CSB-E14993h; both from CUSABIO) according to the manufacturer’s protocols.

Techniques: Clinical Proteomics

Journal: Journal of Translational Medicine

Article Title: Triad of homocysteine, ROCK2 and vimentin as a convergent biomarker signature in pseudoexfoliation

doi: 10.1186/s12967-025-07437-8

Figure Lengend Snippet: Distribution analysis and effect sizes of plasma biomarkers across study groups. ( A–C ) Q–Q plots display the distribution of plasma Hcy, ROCK2, and VIM levels in Control, PEXS, and PEXG groups relative to a theoretical normal distribution. Deviations from the reference line (y = x) indicate non-normality. ( D ) Violin plots show biomarker distribution and effect sizes across group comparisons: Control vs. PEX group, Control vs. PEXS, Control vs. PEXG, and PEXS vs. PEXG. Boxplots indicate medians and interquartile ranges. Cohen’s d values quantify effect sizes. Hcy and VIM showed large effects and consistent upregulation across disease groups, while ROCK2 showed moderate elevation. Minimal separation was observed between PEXS and PEXG for all markers

Article Snippet: Plasma levels of VIM and ROCK2 were measured using commercial ELISA kits (Human Vimentin ELISA Kit, CSB-E08982h; and Human ROCK2 ELISA Kit, CSB-E14993h; both from CUSABIO) according to the manufacturer’s protocols.

Techniques: Clinical Proteomics, Control, Biomarker Discovery

Journal: Journal of Translational Medicine

Article Title: Triad of homocysteine, ROCK2 and vimentin as a convergent biomarker signature in pseudoexfoliation

doi: 10.1186/s12967-025-07437-8

Figure Lengend Snippet: Receiver operating characteristic curves (ROC) of plasma homocysteine, ROCK2, and Vimentin for distinguishing PEXS and PEXG from control subjects. ( A - C ) Hcy showed excellent diagnostic performance with AUCs of 0.98 (95% CI: 0.95–1.00) for Control vs. PEX (PEXS + PEXG), 0.98 (95% CI: 0.95–1.00) for Control vs. PEXS, and 0.97 (95% CI: 0.92–1.00) for Control vs. PEXG. Optimal cut-off values were > 15.09 µmol/L, > 15.19 µmol/L, and > 21.40 µmol/L, respectively. (D-F) ROC analysis of ROCK2 yielded AUCs of 0.77 (95% CI: 0.66–0.88), 0.74 (95% CI: 0.60–0.88), and 0.80 (95% CI: 0.68–0.93) for Control vs. PEX (PEXS + PEXG), PEXS, and PEXG, with corresponding cut-offs of > 375.1 pg/mL, > 378.5 pg/mL, and > 353.1 pg/mL. (G-I) VIM showed AUCs of 0.94 (95% CI: 0.88–0.99), 0.89 (95% CI: 0.79–0.99), and 0.99 (95% CI: 0.97–1.00) for Control vs. PEX (PEXS + PEXG), PEXS, and PEXG, with optimal cut-offs of > 484.60 pg/mL, > 467.30 pg/mL, and > 486.30 pg/mL, respectively

Article Snippet: Plasma levels of VIM and ROCK2 were measured using commercial ELISA kits (Human Vimentin ELISA Kit, CSB-E08982h; and Human ROCK2 ELISA Kit, CSB-E14993h; both from CUSABIO) according to the manufacturer’s protocols.

Techniques: Clinical Proteomics, Control, Diagnostic Assay

Journal: Journal of Translational Medicine

Article Title: Triad of homocysteine, ROCK2 and vimentin as a convergent biomarker signature in pseudoexfoliation

doi: 10.1186/s12967-025-07437-8

Figure Lengend Snippet: Correlation analyses between homocysteine, ROCK2, and Vimentin. ( A ) Global correlation matrix for all the study subjects shows strong positive correlation between expression of Hcy and ROCK2 (ρ = 0.62), ROCK2 and VIM (ρ = 0.69), VIM and Hcy (ρ = 0.79). ( B ) Stratified correlation analysis showing preservation of associations within individual groups with varying strength. ( C ) Dot plot displaying Spearman correlation coefficients (ρ) for each biomarker pair: ROCK2 and VIM, ROCK2 and Hcy, and VIM and Hcy across the three study groups. Strong positive correlations were observed among all pairs in PEXS and PEXG, while correlations in the control group were weak or absent. Notably, the correlation between ROCK2 and VIM increases in PEXS but declines in PEXG. In contrast, the strength of association between Hcy and both ROCK2 and VIM increases progressively from PEXS to PEXG. The dashed line at ρ = 0 indicates no correlation

Article Snippet: Plasma levels of VIM and ROCK2 were measured using commercial ELISA kits (Human Vimentin ELISA Kit, CSB-E08982h; and Human ROCK2 ELISA Kit, CSB-E14993h; both from CUSABIO) according to the manufacturer’s protocols.

Techniques: Expressing, Preserving, Biomarker Discovery, Control

Journal: Journal of Translational Medicine

Article Title: Triad of homocysteine, ROCK2 and vimentin as a convergent biomarker signature in pseudoexfoliation

doi: 10.1186/s12967-025-07437-8

Figure Lengend Snippet: Effect of elevated homocysteine on ROCK2 and VIM in HLE-B3 cells. ( A - B ) Representative immunoblots showing ROCK2 and VIM protein levels in HLE-B3 cells treated with 0 (Vehicle Control), 500 µM, or 1000 µM Hcy for 48 h. GAPDH was used as a loading control. ( C – D ) Quantitative densitometric analysis of ROCK2 ( C ) and VIM ( D ) normalized to GAPDH. Data are presented as mean ± SD from five independent biological replicates ( n = 5). Treatment with Hcy led to a concentration-dependent increase in expression of both markers. Statistical analysis was performed using the Mann–Whitney U test. Significance is indicated as follows: p < 0.05 (*), and p < 0.01 (**)

Article Snippet: Plasma levels of VIM and ROCK2 were measured using commercial ELISA kits (Human Vimentin ELISA Kit, CSB-E08982h; and Human ROCK2 ELISA Kit, CSB-E14993h; both from CUSABIO) according to the manufacturer’s protocols.

Techniques: Western Blot, Control, Concentration Assay, Expressing, MANN-WHITNEY

Journal: Journal of Translational Medicine

Article Title: Triad of homocysteine, ROCK2 and vimentin as a convergent biomarker signature in pseudoexfoliation

doi: 10.1186/s12967-025-07437-8

Figure Lengend Snippet: Diagnostic performance of two-marker and three-marker biomarker models. Receiver operating characteristic (ROC) curves comparing the classification performance of different biomarker combinations for diagnostic accuracy. ( A ) Two-marker model with Hcy and VIM achieved an AUC of 0.98 (95% CI: 0.95–1.00), a cut-off of 0.80, sensitivity of 0.91, and specificity of 0.83. ( B ) Two-marker model with Hcy and ROCK2 showed an AUC of 0.97 (95% CI: 0.94–1.00), with a cut-off of 0.48, sensitivity of 0.94, and specificity of 0.83. ( C ) Two-marker model with VIM and ROCK2 resulted in an AUC of 0.94 (95% CI: 0.88–0.99), a cut-off of 0.47, sensitivity of 0.88, and specificity of 0.80. ( D ) Three-marker model combining Hcy, ROCK2, and VIM yielded comparable diagnostic performance, with an AUC of 0.98 (95% CI: 0.96–1.00), but with a balanced cut-off score of 0.77, sensitivity of 0.94, and specificity of 0.88

Article Snippet: Plasma levels of VIM and ROCK2 were measured using commercial ELISA kits (Human Vimentin ELISA Kit, CSB-E08982h; and Human ROCK2 ELISA Kit, CSB-E14993h; both from CUSABIO) according to the manufacturer’s protocols.

Techniques: Diagnostic Assay, Marker, Biomarker Discovery

Journal: Journal of Translational Medicine

Article Title: Triad of homocysteine, ROCK2 and vimentin as a convergent biomarker signature in pseudoexfoliation

doi: 10.1186/s12967-025-07437-8

Figure Lengend Snippet: Receiver Operating Characteristic (ROC) curves for individual and combined biomarker models distinguishing study groups. ( A ) Control vs. PEX (PEXS + PEXG), ( B ) Control vs. PEXS, ( C ) Control vs. PEXG, ( D ) PEXS vs. PEXG. Each panel shows ROC curves for models based on individual biomarkers: Homocysteine (blue), ROCK2 (orange), Vimentin (green), and a combined LASSO logistic regression model (red). LASSO models were trained using repeated 5-fold cross-validation, and optimism-corrected AUCs were estimated using 1000 bootstrap replicates. AUC values are displayed within each panel

Article Snippet: Plasma levels of VIM and ROCK2 were measured using commercial ELISA kits (Human Vimentin ELISA Kit, CSB-E08982h; and Human ROCK2 ELISA Kit, CSB-E14993h; both from CUSABIO) according to the manufacturer’s protocols.

Techniques: Biomarker Discovery, Control

Journal: Journal of Translational Medicine

Article Title: Triad of homocysteine, ROCK2 and vimentin as a convergent biomarker signature in pseudoexfoliation

doi: 10.1186/s12967-025-07437-8

Figure Lengend Snippet: Calibration and clinical utility of the combined biomarker prediction model. ( A ) Calibration plot for the logistic regression model combining Hcy, ROCK2, and VIM. The x-axis shows predicted disease probability, and the y-axis indicates observed frequency. The navy-blue curve represents the bias-corrected calibration (1000 bootstrap resamples); the diagonal grey dashed line indicates perfect prediction. The model demonstrated good calibration, with a mean absolute error of 0.04 and a mean squared error of 0.003. ( B ) Decision Curve Analysis (DCA) assessing the net clinical benefit of the combined biomarker model. The standardized net benefit is plotted against a range of threshold probabilities. The blue line denotes the biomarker model; the red dashed and gray lines represent the “Treat All” and “Treat None” strategies, respectively. The model shows superior net benefit across the clinically relevant threshold range (0.1–0.8), supporting its potential for individualized, risk-based decision-making

Article Snippet: Plasma levels of VIM and ROCK2 were measured using commercial ELISA kits (Human Vimentin ELISA Kit, CSB-E08982h; and Human ROCK2 ELISA Kit, CSB-E14993h; both from CUSABIO) according to the manufacturer’s protocols.

Techniques: Biomarker Discovery

Journal: Journal of Translational Medicine

Article Title: Triad of homocysteine, ROCK2 and vimentin as a convergent biomarker signature in pseudoexfoliation

doi: 10.1186/s12967-025-07437-8

Figure Lengend Snippet: k-Means (k = 3) clustering of subjects based on biomarker profiles and mean biomarker expression based on z-score. ( A ) PCA plot showing three clusters derived from k-means clustering of standardized Hcy, ROCK2, and VIM values. Clusters show differential enrichment of disease states, with Cluster 3 predominantly composed of controls, Cluster 1 enriched in PEXS, and Cluster 2 enriched in PEXG. Subjects are coloured by disease status and shaped by cluster label. The ellipses represent the 95% confidence boundaries for each cluster. ( B ) Mean biomarker expression (z-score ± SEM) across three unsupervised clusters. Cluster 2 shows coordinated upregulation of Hcy, ROCK2, and VIM, consistent with advanced disease (PEXG). Cluster 3 exhibits downregulated expression typical of controls. Cluster 1 shows mild elevations, suggesting early disease activity (PEXS)

Article Snippet: Plasma levels of VIM and ROCK2 were measured using commercial ELISA kits (Human Vimentin ELISA Kit, CSB-E08982h; and Human ROCK2 ELISA Kit, CSB-E14993h; both from CUSABIO) according to the manufacturer’s protocols.

Techniques: Biomarker Discovery, Expressing, Derivative Assay, Activity Assay

Journal: Journal of Translational Medicine

Article Title: Triad of homocysteine, ROCK2 and vimentin as a convergent biomarker signature in pseudoexfoliation

doi: 10.1186/s12967-025-07437-8

Figure Lengend Snippet: Integrated pathway enrichment analysis of homocysteine, VIM, and ROCK2. ( A ) Bubble plot showing significantly enriched pathways based on –log₁₀(p-value) and pathway impact. Bubble size reflects the number of matched features; colour indicates functional categories. Key pathways highlight redox imbalance, cytoskeletal disruption, and vascular dysfunction. ( B ) Clustered bar plot grouping enriched pathways ( p < 0.05) into functional categories. The most enriched categories were methylation/redox metabolism, cytoskeletal remodelling, and immune signaling, suggesting a central role for metabolic and mechanical stress in PEX pathogenesis

Article Snippet: Plasma levels of VIM and ROCK2 were measured using commercial ELISA kits (Human Vimentin ELISA Kit, CSB-E08982h; and Human ROCK2 ELISA Kit, CSB-E14993h; both from CUSABIO) according to the manufacturer’s protocols.

Techniques: Functional Assay, Disruption, Methylation

Journal: Journal of Translational Medicine

Article Title: Triad of homocysteine, ROCK2 and vimentin as a convergent biomarker signature in pseudoexfoliation

doi: 10.1186/s12967-025-07437-8

Figure Lengend Snippet: STITCH network of PEX-associated genes and homocysteine metabolism. The network includes VIM, ROCK2, their interactors, and homocysteine-metabolizing enzymes. Nodes represent proteins/metabolites; edges indicate functional associations. Distinct modules include cytoskeletal/adhesion (VIM, ROCK1/2, RHOA), methylation/redox (MTHFR, CBS, DNMTs), TGF-β/WNT signaling (TGFB1, ZEB1/2, CTNNB1), and stress response (CASP3, HSPA1A, UBB), illustrating converging pathways in PEX pathogenesis

Article Snippet: Plasma levels of VIM and ROCK2 were measured using commercial ELISA kits (Human Vimentin ELISA Kit, CSB-E08982h; and Human ROCK2 ELISA Kit, CSB-E14993h; both from CUSABIO) according to the manufacturer’s protocols.

Techniques: Functional Assay, Methylation

Journal: Journal of Translational Medicine

Article Title: Triad of homocysteine, ROCK2 and vimentin as a convergent biomarker signature in pseudoexfoliation

doi: 10.1186/s12967-025-07437-8

Figure Lengend Snippet: SP1 as a key transcriptional regulator in Pseudoexfoliation. ( A ) Transcription factor enrichment using TRRUST and ChEA3 identified SP1 as a top-scoring regulator of PEX-associated genes ( B–C ). In silico prediction of putative SP1 binding sites performed within the proximal promoter regions (− 1500 bp upstream of the TSS) of VIM and ROCK2 using the UCSC Genome Browser and JASPAR (motif MA0079.3)

Article Snippet: Plasma levels of VIM and ROCK2 were measured using commercial ELISA kits (Human Vimentin ELISA Kit, CSB-E08982h; and Human ROCK2 ELISA Kit, CSB-E14993h; both from CUSABIO) according to the manufacturer’s protocols.

Techniques: In Silico, Binding Assay