Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Temozolomide increases the generation of cell heterogeneity in ERK activity in glioma cells

doi: 10.1007/s00109-026-02644-2

Figure Lengend Snippet: Phenotypic space of ERK activity in glioblastoma cells. A ERK activity was measured by the cytoplasmic/nuclear (C/N) ratio of the green fluorescence of cells expressing ERK-KTR and 53BP1-Apple. B Four levels of ERK activity were defined as very inactive (VI), inactive (I), active (A), and very active (VA) based on the distributions of ERK activity of A172 cells grown in 10% FBS (orange line) or 0.5% FBS (blue line) for 48 h and C 20 nM trametinib for 2 h (green line) or 2.5 ng/ml of EGF for 15 min (red line) after 48 h of serum deprivation. Relative Shannon Index of 4 groups (rSI4 or SI4 ERK ) calculations are indicated. D Average ERK activity and E phenotypic space (rSI4) occupied by cells treated as in B and C . Each dot represents an image field with at least 20 cells (10% n = 2085 cells; 0.5% n = 4466 cells; TRAM n = 362 cells). F Representative image field of A172 glioma cells and I MRC5 fibroblast cells before (left) and after (right) 15 min of 2.5 ng/ml of EGF treatment (20× magnification). The numbers in the image represent the C/N ratio of each cell. The distribution of cells’ phenotypes and SI4 ERK for this group of cells is shown in the graphs below the images. G Distribution of cells treated with EGF after 48 h of serum deprivation in A172 glioma cells and J MRC5 cells. H SI4 ERK occupied by A172 glioma cells and K MRC5 cells after 15 min of EGF treatment. Each dot represents an image field with at least 20 cells (A172 n = at least 350 cells per EGF dose; MRC5 n = at least 186 cells per EGF dose) One-way ANOVA. EGF, epidermal growth factor; TRAM, trametinib; rSI4 or SI4 ERK , relative Shannon Index of 4 groups of ERK activity); * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: In this study, we used A172 glioblastoma cell line (ATCC, catalog number CRL-1620), U-251 MG glioma cell line (STR validated in October 2018 by the Banco de Células do Rio de Janeiro), U138 MG glioblastoma cell line (ATCC HTB-16), and MRC5 lung fibroblast cell line (ATCC, catalog number CCL-171).

Techniques: Activity Assay, Fluorescence, Expressing

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Temozolomide increases the generation of cell heterogeneity in ERK activity in glioma cells

doi: 10.1007/s00109-026-02644-2

Figure Lengend Snippet: Impact of TMZ on ERK phenotype heterogeneity. A Average and B distribution of ERK activity of A172 cells treated with TMZ (100 mM for 3 h). Cells were imaged every 10 min for 12 h prior to TMZ treatment, during 3 h of treatment, and for 12 h 3 or 10 days after treatment withdrawal. Each dot represents the time-averaged ERK activity of an image field with at least 20 cells (prior to TMZ n = 1834 reads, during TMZ n = 268 reads, 3 days after n = 9683 reads, 10 days after n = 3671 reads). DMSO was used in the same volume as TMZ for control of mechanical ERK stimulation. One-way ANOVA. C Distribution of ERK states frequency prior to TMZ treatment, and 3, 6, or 10 days after treatment withdrawal in U-251 MG cells. D Phenotypic space occupied by A172 or E U-251 MG cells treated as in A . One-way ANOVA. F Average nuclear area of untreated and TMZ treated A172 cells 3, 5, and 10 days after drug removal. Each dot represents the average of a field with at least 30 cells (10× magnification). One-way ANOVA. ** p < 0.005, **** p < 0.0001. G Relation of phenotypic space of ERK activity (SI4 ERK ) and nuclear area heterogeneity (SI4 NucArea ) of A172 glioma cells before TMZ treatment and 3, 5, and 10 days after treatment withdrawal. Each dot represents the SI4 ERK /SI4 NucArea of a field with at least 30 cells (10× magnification). Unpaired t -test. H Phenotypic space of ERK activity occupied by the cells of A172 colonies immediately before (day 4) and 3 days after treatment withdrawal (day 7). Each dot represents the SI4 ERK of a colony of cells imaged every 10 min for 3 h (10× magnification) (untreated n = 32; TMZ treated n = 26). Unpaired t -test. I Change in SI4 ERK of each colony after TMZ treatment and its relationship with colony growth. Each dot represents the SI4 ERK of a colony of cells imaged every 10 min for 3 h (10× magnification). Arrows indicate the pathway from initial to final SI4 ERK and colony size of each colony. Blue dots indicate homogeneous colonies, and red dots indicate heterogeneous colonies. J Correlations among colony size (CS), average ERK activity of the colony (AV ERK), and phenotypic space of ERK activity (SI4 ERK ), together with the deltas (∆CS, ∆AV ERK , and ∆SI4 ERK ) of these features from day 4 (B, before TMZ) and day 7 (A, 3 days after TMZ withdrawal). Relevant positive and negative correlations are shown in detail ( J (a, b, c, d))

Article Snippet: In this study, we used A172 glioblastoma cell line (ATCC, catalog number CRL-1620), U-251 MG glioma cell line (STR validated in October 2018 by the Banco de Células do Rio de Janeiro), U138 MG glioblastoma cell line (ATCC HTB-16), and MRC5 lung fibroblast cell line (ATCC, catalog number CCL-171).

Techniques: Activity Assay, Control

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Temozolomide increases the generation of cell heterogeneity in ERK activity in glioma cells

doi: 10.1007/s00109-026-02644-2

Figure Lengend Snippet: Reduction of ERK phenotype heterogeneity reduces fractional killing of clonal populations. A Average of ERK activity from cells treated with TMZ (100 mM for 3 h); TRAM (20 nM for 24 h) or a combination of TMZ and TRAM. Cells were imaged every 10 min for 12 h prior to TMZ treatment, during 3 h of treatment and for 12 h 3 or 10 days after treatment withdrawal. Each dot represents the time-averaged ERK activity of an image field with at least 20 cells (TMZ during n = 268, TMZ 3 d n = 9683, TMZ 10 d n = 3671; TRAM during n = 362; TRAM 3 d n = 331; TRAM 10 d n = 245; TMZ + TRAM during n = 428, TMZ + TRAM 3 d n = 650; TMZ + TRAM 10 d n = 599). One-way ANOVA. B Impact of MEK inhibition with TRAM on the SI4 ERK 3 or 10 days after drug withdrawal in A172 and C U-251 MG cells. Cells were treated as in A . One-way ANOVA. D Colony size of A172 cells for each treatment type on day 14. Each dot represents a colony. Unpaired, two-sided Mann–Whitney U test. E Lethal fraction (LF) over time of A172 colonies after TMZ, F TRAM or G TMZ and TRAM treatments. Heterogeneity in LF induction was calculated as the Shannon Index diversity (SI4 LF ) for 4 equal categories. Black lines represent the average of LF of all colonies. H LF of colonies whose phenotypic heterogeneity remained stable (stb), increased (inc) or decreased (dec) 3 days after TMZ treatment. ∆SI4 ERK was calculated as SI4 ERK after TMZ treatment minus SI4 ERK before treatment. ∆SI4 ERK was considered stable when it changed to less than 10%. One-way ANOVA. I Maximum LF observed after TMZ, TRAM, or TMZ and TRAM treatment. Each dot represents a colony (untreated n = 37; TMZ n = 64; TRAM n = 39; TMZ and TRAM n = 33). J Proportion of colonies with > 75%, 25–75% or < 25% of death rate at the end of the experiment for TMZ (above) and TMZ and TRAM treatments (lower). LF (lethal fraction); TMZ (temozolomide); TRAM (trametinib); ns, non-significative; * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.001

Article Snippet: In this study, we used A172 glioblastoma cell line (ATCC, catalog number CRL-1620), U-251 MG glioma cell line (STR validated in October 2018 by the Banco de Células do Rio de Janeiro), U138 MG glioblastoma cell line (ATCC HTB-16), and MRC5 lung fibroblast cell line (ATCC, catalog number CCL-171).

Techniques: Activity Assay, Inhibition, MANN-WHITNEY

Journal: CNS Neuroscience & Therapeutics

Article Title: The HBP Pathway Inhibitor FR054 Enhances Temozolomide Sensitivity in Glioblastoma Cells by Promoting Ferroptosis and Inhibiting O‐ GlcNAcylation

doi: 10.1111/cns.70546

Figure Lengend Snippet: Proteomics analysis reveals enrichment of the hexosamine biosynthesis pathway and upregulation of O‐GlcNAcylation in temozolomide‐resistant cells. (A) U87‐MG and A172 cells were subjected to gradually increasing TMZ concentrations over approximately 10 weeks to induce resistance. (B) The IC50 values of TMZ‐resistant strains were approximately two‐fold higher than those of parental cells, confirming significant acquired resistance. (C) Schematic workflow of protein extraction and mass spectrometry analysis performed on TMZ‐resistant strains and GBM cell lines. (D) Volcano plot of differential protein expression. Proteins with fold change (FC) > 1.5 and p < 0.05 were defined as upregulated; FC < 0.67 and p < 0.05 as downregulated. (E) Bar plots showing the top 10 enriched GO‐BP and KEGG pathways for upregulated genes in U87‐TR vs. U87 (left) and A172‐TR vs. A172 (right), ranked by enrichment scores. Color intensity reflects statistical significance ( p ‐value). (F) Schematic representation of the hexosamine biosynthetic pathway (HBP), illustrating the enzymatic conversion of glucose and glutamine into UDP‐GlcNAc. (G) Western blot analysis of O‐GlcNAc expression in U87 and U87‐TR cells with or without 72‐h stimulation by 400 μM TMZ. (H) Western blot analysis of O‐GlcNAc expression in A172 and A172‐TR cells with or without 72‐h stimulation by 400 μM TMZ.

Article Snippet: The U87‐MG and A172 cell lines used in this study were purchased from the American Type Culture Collection (ATCC).

Techniques: Protein Extraction, Mass Spectrometry, Expressing, Western Blot

Journal: CNS Neuroscience & Therapeutics

Article Title: The HBP Pathway Inhibitor FR054 Enhances Temozolomide Sensitivity in Glioblastoma Cells by Promoting Ferroptosis and Inhibiting O‐ GlcNAcylation

doi: 10.1111/cns.70546

Figure Lengend Snippet: Combination therapy of TMZ and HBP Pathway Inhibitor FR054 displayed a significant synergistic effect on GBM cells. (A) Molecular docking of FR054 with PGM3 protein. Generated using CB‐Dock2, this model visualizes the potential binding sites of FR054 (spherical structures) to the PGM3 protein (in blue), providing insights into their interaction at the molecular level. (B) CCK8 assay IC50 curves for U87, U87‐TR, A172, A172‐TR, and GBM001 cells following 72‐h treatment with FR054. (C) Colony formation assay of U87, U87‐TR, A172, and A172‐TR cells after 14‐day drug treatment. Left: Representative colony images. Right: Quantitative analysis of colony numbers, normalized to untreated controls. (D) Heatmap of Bliss synergy indices for U87, U87‐TR, A172, and A172‐TR cells treated with varying concentrations of TMZ and FR054 for 72 h, calculated using SynergyFinder. Higher synergy scores indicate stronger synergistic effects. (E) OD450 values for U87, U87‐TR, A172, and A172‐TR cells treated with TMZ, FR054, or their combination for 72 h. Bliss index‐optimized concentrations were used to evaluate cell viability. Statistical comparisons were performed using Student's t‐test at 72 h. (F) IC50 curves of U87, U87‐TR, A172, and A172‐TR cells treated with FR054 in combination with TMZ at varying concentrations, assessed by CCK8 assay. These curves demonstrate the dose‐dependent reduction in TMZ IC50 values upon FR054 co‐treatment.

Article Snippet: The U87‐MG and A172 cell lines used in this study were purchased from the American Type Culture Collection (ATCC).

Techniques: Generated, Binding Assay, CCK-8 Assay, Colony Assay

Journal: CNS Neuroscience & Therapeutics

Article Title: The HBP Pathway Inhibitor FR054 Enhances Temozolomide Sensitivity in Glioblastoma Cells by Promoting Ferroptosis and Inhibiting O‐ GlcNAcylation

doi: 10.1111/cns.70546

Figure Lengend Snippet: Synergistic anti‐tumor effects of FR054 and TMZ on glioblastoma cells through downregulating protein O‐GlcNAcylation and activating ferroptosis. (A) Western blot analysis of O‐GlcNAc levels in U87, U87‐TR, A172, and A172‐TR cells after 72‐h treatments with TMZ, FR054, or their combination. (B) Transcriptional profiling of U87‐TR cells treated for 72 h with DMSO, TMZ, FR054, or TMZ + FR054; RNA sequencing was performed to identify differentially expressed genes (DEGs). (C) Volcano plots illustrating differentially expressed genes (DEGs) between the TMZ + FR054 vs. TMZ and FR054 vs. DMSO groups (|log2FC| > 1, P adj < 0.05). (D) Bar graph depicting KEGG pathway enrichment analysis for the FR054 vs. DMSO comparison, ordered by statistical significance. (E) Gene Set Enrichment Analysis (GSEA) focusing on ferroptosis‐ and ROS‐related pathways for upregulated genes in the TMZ + FR054 vs. TMZ and FR054 vs. DMSO groups. (F) Heatmap displaying the expression levels of ferroptosis‐promoting genes across four experimental groups.

Article Snippet: The U87‐MG and A172 cell lines used in this study were purchased from the American Type Culture Collection (ATCC).

Techniques: Western Blot, RNA Sequencing, Comparison, Expressing