Journal: Pharmaceuticals

Article Title: Identification of Methylstat as a Potential Therapeutic Agent for Human Glioma Cells by Targeting Cell Cycle Arrest

doi: 10.3390/ph18091344

Figure Lengend Snippet: Comprehensive Analysis of Transcriptome Sequencing and Connectivity Map in Human Glioma Cells. ( a ) Network analysis of gene co-expression patterns in TB-treated HOG and U251 glioma cells using the Connectivity Map database. Five small-molecule compounds showing significant positive correlations are identified; ( b ) Volcano plots depicting differential gene expression (DEGs) in TB-treated glioma cells. Black: genes that are not significantly deregulated; green: downregulated genes; red, upregulated genes; ( c ) KEGG enrichment analysis of differentially expressed genes in HOG and U251 cells. Bubble size corresponds to the number of enriched genes per pathway, while color intensity represents statistical significance (−log 10 ( p -value)); ( d ) The chemical structures of topotecan, importazole, NSC-663284, methylstat, and ryuvidine; ( e ) Cell viability assessment by CCK-8 assay in glioma cells treated with methylstat (0–6 μM for U251, 0–4 μM for HOG) for 24, 48, and 72 h. ( f ) Differential sensitivity to methylstat exposure (48-h treatment) across glioma cell lines (HOG, U251) versus primary cultured astrocytes. Values are presented as mean ± SD ( n = 3).

Article Snippet: Methylstat (cat: HY-1522, MCE, Shanghai, China) was dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA) to prepare a 20 mM stock solution, stored at −20 °C.

Techniques: Sequencing, Expressing, Gene Expression, CCK-8 Assay, Cell Culture

Journal: Pharmaceuticals

Article Title: Identification of Methylstat as a Potential Therapeutic Agent for Human Glioma Cells by Targeting Cell Cycle Arrest

doi: 10.3390/ph18091344

Figure Lengend Snippet: Morphological Changes and Nuclear Staining in Methylstat-Treated Glioma Cells. ( a ) Morphological changes and nuclear staining (DAPI) of U251 glioma cells after treatment with varying concentrations (0–6 μM) of methylstat for 48 h. The triangular gradient overlay indicates the methylstat concentration gradient (0 μM to 6 μM); ( b ) Morphological changes and DAPI of HOG glioma cells treated with varying concentrations (0–4 μM) of methylstat for 48 h. The triangular gradient overlay indicates the methylstat concentration gradient (0 μM to 4 μM);. Scale bar: 50 μm.

Article Snippet: Methylstat (cat: HY-1522, MCE, Shanghai, China) was dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA) to prepare a 20 mM stock solution, stored at −20 °C.

Techniques: Staining, Concentration Assay

Journal: Pharmaceuticals

Article Title: Identification of Methylstat as a Potential Therapeutic Agent for Human Glioma Cells by Targeting Cell Cycle Arrest

doi: 10.3390/ph18091344

Figure Lengend Snippet: Assessment of Apoptosis in Methylstat-Treated Glioma Cells. ( a ) TUNEL/DAPI staining of U251 cells treated with methylstat (0–6 μM, 48 h); ( b ) TUNEL/DAPI staining of HOG cells treated with methylstat (0–4 μM, 48 h); ( c ) Annexin V/PI flow cytometry analysis of U251 (4–6 μM) and HOG (2–4 μM) cells demonstrates no statistically significant elevation in early (Annexin V+/PI−) or late (Annexin V+/PI+) apoptotic populations compared to untreated controls (mean ± SD, n = 3). Scale bar: 100 μm.

Article Snippet: Methylstat (cat: HY-1522, MCE, Shanghai, China) was dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA) to prepare a 20 mM stock solution, stored at −20 °C.

Techniques: TUNEL Assay, Staining, Flow Cytometry

Journal: Pharmaceuticals

Article Title: Identification of Methylstat as a Potential Therapeutic Agent for Human Glioma Cells by Targeting Cell Cycle Arrest

doi: 10.3390/ph18091344

Figure Lengend Snippet: Methylstat Treatment Inhibit Glioma Cell Proliferation in vitro. ( a ) Ki67/EdU immunofluorescence staining in U251 and HOG cells treated with methylstat for 48 h. A significant reduction in Ki67-positive (green) and EdU-positive (red) cells indicates decreased proliferation; ( b ) Quantitative analysis reveals that methylstat treatment reduces the percentage of proliferating cells; ( c ) Colony formation assays (5000 cells/well seeded) show a dose-dependent decrease in colony counts following 10 days continuous methylstat treatment in both cell lines.

Article Snippet: Methylstat (cat: HY-1522, MCE, Shanghai, China) was dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA) to prepare a 20 mM stock solution, stored at −20 °C.

Techniques: In Vitro, Immunofluorescence, Staining

Journal: Pharmaceuticals

Article Title: Identification of Methylstat as a Potential Therapeutic Agent for Human Glioma Cells by Targeting Cell Cycle Arrest

doi: 10.3390/ph18091344

Figure Lengend Snippet: Effects of Methylstat on Cell Cycle Regulation in Glioma Cells. ( a ) Flow cytometry analysis of U251 cells treated with methylstat (0–6 μM, 48 h) reveals G1 phase accumulation. Red: G1 phase; Blue: S phase; Pink: G2 phase; ( b ) Quantitative assessment of cell cycle distribution in U251 cells after methylstat treatment; ( c ) qPCR analysis in U251 cells indicates downregulation of cyclin D , cyclin A , CDK2 , and CDK4 , with upregulation of p 21 upon methylstat treatment. Values are presented as mean ± SD of triplicate; ( d ) Flow cytometry of HOG cells treated with methylstat (0 to 4 μM, 48 h) shows an increase in G2 phase arrest. Red: G1 phase; Blue: S phase; Pink: G2/M phase; ( e ) Quantitative assessment of cell cycle distribution in HOG cells after methylstat treatment; ( f ) In HOG cells, qPCR shows reduced expression of cyclin B , CDK1 , and CDC25C , but enhanced p53 and p21 levels post-treatment. Values are presented as mean ± SD of triplicate; ( g ) Western blot analyses of the expression levels of p21, p53, CDK4, cyclin A, cyclin D, CDK1, CDC25C, Cyclin B from U251 cells or HOG cells treated with methylstat, where GAPDH and β-actin were used as loading controls for U251 and HOG cells, respectively. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns, not significant (Student’s t -test).

Article Snippet: Methylstat (cat: HY-1522, MCE, Shanghai, China) was dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA) to prepare a 20 mM stock solution, stored at −20 °C.

Techniques: Flow Cytometry, Expressing, Western Blot

Journal: Pharmaceuticals

Article Title: Identification of Methylstat as a Potential Therapeutic Agent for Human Glioma Cells by Targeting Cell Cycle Arrest

doi: 10.3390/ph18091344

Figure Lengend Snippet: Effects of Methylstat on JMJD2A Expression and Enzymatic Activity in Glioma Cells. ( a ) qPCR analysis of U251 cells treated with methylstat (0–6 μM, 48 h) shows significant reductions in JMJD2A , PDK1 , AKT , and mTOR mRNA levels. Values are presented as mean ± SD of triplicate experiments; ( b ) qPCR analysis of HOG cells treated with methylstat (0–4 μM, 48 h) reveals a marked decrease in JMJD2A , PDK1 , AKT , and mTOR mRNA levels. Values are presented as mean ± SD of triplicate experiments; ( c ) 3D structure modeling, based on the JMJD2A crystal structure (PDB 2P5B) and Methylstat (PubChem 53392493), reveals stable binding of methylstat within the active site of JMJD2A; ( d ) Root mean square deviation (RMSD) profile of the JMJD2A–methylstat complex over 200 ns of molecular dynamics (MD) simulation; ( e ) Radius of gyration (Rg) trajectory during the MD simulation, reflecting structural compactness; ( f ) Solvent-accessible surface area (SASA) of the complex throughout the simulation, indicating conformational stability; ( g ) Root mean square fluctuation (RMSF) analysis comparing backbone flexibility of JMJD2A in apo and holo states; ( h ) Effect of methylstat on JMJD2A enzyme activity of glioma cells. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns, not significant (Student’s t -test).

Article Snippet: Methylstat (cat: HY-1522, MCE, Shanghai, China) was dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA) to prepare a 20 mM stock solution, stored at −20 °C.

Techniques: Expressing, Activity Assay, Binding Assay, Solvent

Journal: Pharmaceuticals

Article Title: Identification of Methylstat as a Potential Therapeutic Agent for Human Glioma Cells by Targeting Cell Cycle Arrest

doi: 10.3390/ph18091344

Figure Lengend Snippet: Schematic Diagram of Methylstat-Induced Cell Cycle Arrest and its Regulatory Mechanisms in Glioma Cells. Methylstat differentially regulates glioma cell cycle progression via two distinct pathways: in HOG cells, it induces G2/M arrest through a p53 -dependent cascade ( p53 activation → p21 upregulation → downregulation of CDC25C / CDK1 / cyclin B → G2/M transition blockade), whereas in U251 cells, methylstat causes G1 accumulation via p21 -mediated inhibition of the cyclin D-CDK4 complex (p21 elevation → disruption of cyclin D-CDK4 assembly → G1/S transition suppression); these differential effects are synergistically enhanced by methylstat-driven suppression of JMJD2A and attenuation of the PDK1/AKT/mTOR pro-proliferative signaling pathway, ultimately resulting in collective inhibition of glioma cell proliferation through dual-phase (G1 and G2/M) cell cycle arrest.

Article Snippet: Methylstat (cat: HY-1522, MCE, Shanghai, China) was dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA) to prepare a 20 mM stock solution, stored at −20 °C.

Techniques: Activation Assay, Inhibition, Disruption

Journal: Cancer Gene Therapy

Article Title: Methylstat sensitizes ovarian cancer cells to PARP-inhibition by targeting the histone demethylases JMJD1B/C

doi: 10.1038/s41417-025-00874-z

Figure Lengend Snippet: Pharmacological inhibitors screened for synergistic interactions with olaparib.

Article Snippet: Methylstat (#SML0343; selective inhibitor of Jumonji C domain-containing histone demethylases, including JMJD1B, JMJD1C) [ ] was purchased from Sigma Aldrich and Metformin (#317240-5 G, target: SP1) [ ] from EMD Millipore.

Techniques:

Journal: Cancer Gene Therapy

Article Title: Methylstat sensitizes ovarian cancer cells to PARP-inhibition by targeting the histone demethylases JMJD1B/C

doi: 10.1038/s41417-025-00874-z

Figure Lengend Snippet: A Drug interaction analysis of methylstat and olaparib according to the combination index (CI) method. Drugs were combined at a broad range of equipotent molar concentrations, according to the indicated IC 100 , IC 50 , IC 25 , IC 12.5 , IC 6,25 . Standard deviation from three independent experiments is indicated by error bars. Resulting CI-values are indicated in the table. A drug interaction with a CI-value < 1.0 is classified as synergistic (bold numbers). B Drug interaction analysis of methylstat and olaparib according to Bliss synergy score calculation. A score >10 indicates synergy. Results were derived from three independent biological replicates. Kaplan-Meier analysis showing prognostic relevance of ( C ) JMJD1B and ( D ) JMJD1C according to the publicly available Kaplan-Meier plotter tool. PFS progression-free survival, OS overall survival.

Article Snippet: Methylstat (#SML0343; selective inhibitor of Jumonji C domain-containing histone demethylases, including JMJD1B, JMJD1C) [ ] was purchased from Sigma Aldrich and Metformin (#317240-5 G, target: SP1) [ ] from EMD Millipore.

Techniques: Standard Deviation, Derivative Assay

Journal: Cancer Gene Therapy

Article Title: Methylstat sensitizes ovarian cancer cells to PARP-inhibition by targeting the histone demethylases JMJD1B/C

doi: 10.1038/s41417-025-00874-z

Figure Lengend Snippet: Drug interaction analysis of methylstat and olaparib according to the combination index (CI) method with the indicated cell lines ( A – H ). Drugs were combined at a broad range of equipotent molar concentrations, according to the indicated IC 100 , IC 50 , IC 25 , IC 12.5 , IC 6,25 . Standard deviation from three independent experiments is indicated by error bars. Resulting CI-values are indicated in the table. A drug interaction with a CI-value < 1.0 is classified as synergistic (bold values). Results were derived from three independent biological replicates.

Article Snippet: Methylstat (#SML0343; selective inhibitor of Jumonji C domain-containing histone demethylases, including JMJD1B, JMJD1C) [ ] was purchased from Sigma Aldrich and Metformin (#317240-5 G, target: SP1) [ ] from EMD Millipore.

Techniques: Standard Deviation, Derivative Assay

Journal: Cancer Gene Therapy

Article Title: Methylstat sensitizes ovarian cancer cells to PARP-inhibition by targeting the histone demethylases JMJD1B/C

doi: 10.1038/s41417-025-00874-z

Figure Lengend Snippet: A Schematic overview of a dual-fluorescence clonal competition model. Part of the scheme has been created with Biorender.com. Fluorescent microscopy images of ( B ) PARPi-resistant Olres-UWB1.289-EGFP vs . PARPi sensitive UWB1.289-tdTomato and ( C ) BRCA1 -deficient UWB1.289-tdTomato vs. BRCA1 -proficient UWB1.289+BRCA1-EGFP cell lines, co-cultured in the presence of drug-free medium, olaparib or methylstat+olaparib. P-values according to the one-way ANOVA with post-hoc Tukey HSD test are indicated; * padj < 0.05; ** padj < 0.01; *** padj < 0.001. Results were derived from three independent biological replicates.

Article Snippet: Methylstat (#SML0343; selective inhibitor of Jumonji C domain-containing histone demethylases, including JMJD1B, JMJD1C) [ ] was purchased from Sigma Aldrich and Metformin (#317240-5 G, target: SP1) [ ] from EMD Millipore.

Techniques: Fluorescence, Microscopy, Cell Culture, Derivative Assay