human gbm cell lines u87 mg u87 mg  (ATCC)

Journal: bioRxiv

Article Title: Multi-omics and spatial analysis of microgravity-grown glioblastoma organoids reveals superior modeling of advanced disease after long-term spaceflight

doi: 10.64898/2026.03.06.710192

Figure Lengend Snippet: ( A ) Stacked bar plots summarize the number of significantly DEGs identified by DESeq2 (Benjamini-Hochberg corrected p < 0.05) for four contrasts derived from a model including gravity condition (uG vs KSC), co-culture condition (U87 GBM with and without THP-1 monocytes), and their interaction. Bars show DEGs for (i) U87 (uG vs KSC), (ii) U87THP (uG vs KSC), (iii) uG (U87THP vs U87), and (iv) KSC (U87THP vs U87). Counts are partitioned into upregulated (positive log2FC; light red) and downregulated (negative log2FC; light blue) genes, with the number of DEGs shown. An adjacent UpSet plot displays intersections among DESeq2-identified DEGs. Vertical bars indicate the number of genes in each intersection, and filled dots denote the contrasts contributing to each intersection. ( B ) Volcano plot shows DESeq2 results for the gravity * co-culture interaction term from a DESeq2 model including gravity condition (uG vs KSC), co-culture composition (U87 GBM with and without THP-1 monocytes), and their interaction. Each point represents a gene, plotted by the interaction log2 fold-change (x-axis; difference in the microgravity effect between U87THP and U87) and -log10 adjusted p-value (y-axis; Benjamini-Hochberg corrected). Genes with p < 0.05 are highlighted as significant (red/blue by direction), while non-significant genes are shown in gray; shaded regions indicate the significance threshold. All significant interaction genes are labeled. ( C ) Dot plots show DESeq2 log2 fold-change (uG vs KSC) estimates for selected genes (FAM50A, PRSS35, EPHA7, LINC01705) in U87 GBM alone and GBM co-cultured with THP-1 monocytes. Points indicate the estimated log2FC for each contrast, and error bars represent ±1 standard error (lfcSE). Lines connecting the two contrasts are shown to emphasize differences in direction and magnitude of the microgravity response depending on immune context. A dashed horizontal line marks no change (log2FC = 0). ( D ) GSEA of U87 GBM differentially expressed genes (uG vs KSC) was performed using GBM meta-programs (MPs) . Enrichment plot is shown for MES_2. Running enrichment score (ES) is plotted across the ranked gene list (top), with tick marks indicating positions of MES_2 genes in the ranked list (middle), and the underlying ranking metric shown below (bottom). The annotated inset reports the normalized enrichment score (NES) and Benjamini-Hochberg corrected p-value for MES_2 MP.

Article Snippet: Human GBM U87 cells and human monocyte THP-1 cells were purchased from ATCC (#HTB-14 and #TIB-202).

Techniques: Derivative Assay, Co-Culture Assay, Labeling, Cell Culture

Journal: bioRxiv

Article Title: Multi-omics and spatial analysis of microgravity-grown glioblastoma organoids reveals superior modeling of advanced disease after long-term spaceflight

doi: 10.64898/2026.03.06.710192

Figure Lengend Snippet: ( A ) Volcano plots show DESeq2 statistics comparing microgravity-grown organoids to Kennedy Space Center (KSC) 1 g controls for U87 GBM alone ( A ) and GBM co-cultured with THP-1 monocytes ( B ). Each point represents a gene, plotted by log2 fold change (x-axis; microgravity vs control) and -log10 adjusted p-value (y-axis; Benjamini-Hochberg corrected). Genes meeting p < 0.05 are highlighted as significantly upregulated (red) or downregulated (blue); non-significant genes are shown in gray. Shaded regions indicate the significance threshold (P < 0.05; fold-change threshold set to 0), and the top 20 most significant differentially expressed genes in each comparison are labeled. ( C ) Bar plots show Gene Ontology (GO) over-representation analysis (ORA) of genes differentially expressed in microgravity-grown organoids relative to KSC 1 g controls. Results are shown separately for U87 GBM alone ( C ) and U87 GBM co-cultured with THP-1 monocytes ( D ), with downregulated (blue; left-extending bars) and upregulated (red; right-extending bars) gene sets displayed in separate panels. Bars represent fold enrichment for selected GO terms, and color intensity encodes statistical significance (-log10 adjusted p-value, Benjamini-Hochberg correction). GO terms were simplified to reduce redundancy, and a curated subset of biologically relevant terms is shown for clarity.

Article Snippet: Human GBM U87 cells and human monocyte THP-1 cells were purchased from ATCC (#HTB-14 and #TIB-202).

Techniques: Cell Culture, Control, Comparison, Labeling

Journal: bioRxiv

Article Title: Multi-omics and spatial analysis of microgravity-grown glioblastoma organoids reveals superior modeling of advanced disease after long-term spaceflight

doi: 10.64898/2026.03.06.710192

Figure Lengend Snippet: ( A ) Gene set enrichment analysis (GSEA) was performed using GBM meta-program (MP) gene sets and Wald test DESeq2 statistic for U87 organoids grown in microgravity relative to KSC 1 g controls. Enrichment plots are shown for MP_1_RP (ribosomal protein program), MP_5_Hypoxia (hypoxia response), and MP_15_Stress2 (stress response). In each panel, the running enrichment score (ES) is plotted across the ranked gene list (top), with tick marks indicating positions of MP genes in the ranked list (middle), and the underlying ranking metric shown below (bottom). The annotated inset reports the normalized enrichment score (NES) and Benjamini-Hochberg corrected p-value for each meta-program. ( B ) Heatmap shows leading-edge genes (i.e. core enrichment genes) from GSEA presented in ( A ). Rows correspond to leading-edge genes, and columns represent individual samples annotated by experimental condition (top bar). The main heatmap displays mean-centered and scaled variance-stabilized expression values from DESeq2. The adjacent heatmap (“LFC”) shows the corresponding DESeq2 log2 fold change (microgravity vs control) for each leading-edge gene, with color indicating effect direction and magnitude. (C) GSEA of GBM meta-programs (MPs) was performed as in ( A ), but using DESeq2-ranked statistics from U87 organoids co-cultured with THP-1 monocytes grown in microgravity relative to KSC 1 g controls. (D) GSEA was performed on U87 organoids co-cultured with THP-1 monocytes using MSigDB Hallmark gene sets, with genes ranked by the DESeq2 Wald test statistic. ( E ) Scatter plots summarize HOMER motif enrichment results, with motifs ordered by significance (x-axis, rank) and plotted by motif enrichment log p-value (y-axis; more significant motifs appear higher due to reversed y-scale). Known motif enrichment results are shown for downregulated genes in U87 GBM (left) and upregulated genes in U87 + THP-1 monocyte co-culture (right). Each point represents one annotated HOMER motif; motifs passing significance threshold (P < 0.01, shaded region) are highlighted, and select motifs are labeled. ( F ) Density plot of HOMER-called motif offsets relative to the transcription start site (TSS) across genes used for enrichment analysis in ( I ); the dashed vertical line marks the TSS (0 bp). ( G ) Sequences logos and enrichment statistics are shown for significantly enriched motifs, based on analysis shown in ( E ). Gene Ontology (GO) over-representation analysis (ORA) was performed on downregulated and upregulated genes in U87 ( H ) and U87 + THP-1 monocyte ( I ) organoids, respectively, whose regulatory regions contained at least one TEAD1/2 (in U87; panel H ) or Smad3 (U87+THP-1; panel I ) motif match. Enriched GO terms were simplified to reduce redundancy. Bar plots display selected Biological Process terms (top terms by significance), with bar length indicating fold enrichment and color encoding statistical significance (-log10 p-value, Benjamini-Hochberg corrected). ( J ) Schematic demonstrating likely mechanism by which microgravity impacts GBM phenotype. Under normal gravity or mechanical force, RhoA promotes F-actin polymerization, facilitating YAP/TAZ nuclear localization and interaction with TEAD transcription factors to drive mesenchymal (MES) gene expression. In microgravity conditions, reduced mechanical loading disrupts cytoskeletal tension, leading to inhibition of YAP/TAZ activation and decreased transcription of MES-associated genes.

Article Snippet: Human GBM U87 cells and human monocyte THP-1 cells were purchased from ATCC (#HTB-14 and #TIB-202).

Techniques: Expressing, Control, Cell Culture, Co-Culture Assay, Labeling, Gene Expression, Inhibition, Activation Assay

Journal: bioRxiv

Article Title: Multi-omics and spatial analysis of microgravity-grown glioblastoma organoids reveals superior modeling of advanced disease after long-term spaceflight

doi: 10.64898/2026.03.06.710192

Figure Lengend Snippet: ( A ) Uniform Manifold Approximation and Projection (UMAP) embedding of the scRNA-seq reference dataset combining U87 GBM cells and THP-1 monocytes. Cells are colored by cell type. ( B ) Heatmap of marker gene expression in the scRNA-seq reference dataset. Marker sets include myeloid/innate immune genes (e.g., TYROBP, PTPRC, FCER1G, AIF1, APOE) and mesenchymal-like GBM genes (e.g., ALDH1A3, SERPINE1, TNC, SPARC, LOXL2). Genes were selected as markers identified by Wilcoxon rank-sum test and combined into a curated, nonredundant list for visualization. ( C ) Scatterplot of cell-type weights inferred by robust cell type decomposition (RCTD). Each point represents one cell, plotted by its inferred THP-1 weight (x-axis) and U87 weight (y-axis) from the RCTD weights matrix. The dashed diagonal indicates equal weights (y = x). ( D ) Dot plots summarizing the fraction of cells expressing canonical monocyte markers (CD14, FCN1, CCR2, CSF1R, FCGR1A, FCGR3A, ITGAM, ITGB2, SPI1, IRF8, SIRPA, CLEC12A, BST1). Left: scRNA-seq reference (U87 + THP-1). Right: Xenium single-cell dataset.

Article Snippet: Human GBM U87 cells and human monocyte THP-1 cells were purchased from ATCC (#HTB-14 and #TIB-202).

Techniques: Marker, Gene Expression, Expressing, Single Cell

Journal: MedComm

Article Title: Integrative Single‐Cell Analysis Reveals Targetable Vacuole Membrane Protein 1‐Mediated Mechanism of Tumor Angiogenesis in Glioblastoma

doi: 10.1002/mco2.70619

Figure Lengend Snippet: Overexpression of VMP1 promotes tumor growth. (A) Representative western blot image validating exogenous overexpression of VMP1 in U87 and U251 cell lines. (B) U87 subcutaneous xenografts of overexpressed VMP1 (VMP1‐OE) and control vector at Day 17 post‐injection ( n = 9). (C) The weight of the tumor grafts. (D) Quantification of normalized tumor weights of VMP1‐OE and vector (measured every 4 days from Day 3) ( n = 9). (E) Bioluminescence imaging of U87 VMP1‐OE and vector orthotopic xenografts at different time points from Day 3 to Day 17 ( n = 6). (F) Representative images of hematoxylin and eosin‐stained sections at Day 17 post‐injection. Tumor is indicated within the dashed line. Scale bar, 1000 µm. (G) Relative total photon flux of bioluminescence in mice with U87 VMP1‐OE and vector. (H) Kaplan–Meier survival analysis of mice with U87 VMP1‐OE and vector intracranial xenografts ( n = 6). (I) Top, immunohistochemical staining showing Ki67‐positive cells in subcutaneous and intracranial xenografts. Scale bar, 100 µm. Bottom, quantification of Ki67‐positive cells (%) in subcutaneous and intracranial models. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Human embryonic kidney cells 293T (293T) and human GBM cell lines U87 and U251 were purchased from the American Type Culture Collection (ATCC).

Techniques: Over Expression, Western Blot, Control, Plasmid Preparation, Injection, Imaging, Staining, Immunohistochemical staining

Journal: MedComm

Article Title: Integrative Single‐Cell Analysis Reveals Targetable Vacuole Membrane Protein 1‐Mediated Mechanism of Tumor Angiogenesis in Glioblastoma

doi: 10.1002/mco2.70619

Figure Lengend Snippet: VMP1 promoted tumor growth was independent of autophagy. (A) Representative western blot images of autophagy markers (p62 and LC3 I/II) in U87 and U251 cell lines with VMP1‐OE. (B) Representative transmission electron microscopy images of a cell in U87 and U251 with VMP1‐OE, showing no differences in autophagosome formation. Top: scale bar, 2 µm. Bottom: scale bar, 500 nm. (C) Western blot images of tissue samples from our glioma cohort (glioma) ( n = 47) and normal brain tissue (N), showing the protein expression of autophagy markers (p62, Beclin 1, and LC3 I/II). (D) Quantification of western blot images, patients were separated into two groups based on median VMP1 expression: VMP1 low glioma ( n = 23) and VMP1 high glioma ( n = 24). (E) Correlation analysis of western blot quantification value between VMP1 and autophagy markers (p62, Beclin 1, and LC3 I/II). (F) Confirmation of VMP1 knockdown in U87 and U251 cells using two different targeting sequences by western blot analysis. (G) U87 subcutaneous xenografts of VMP1 knockdown (shVMP1) and control vector (shNC) at Day 27 post‐injection ( n = 10) (left), and the tumor volume measured from Day 14 to Day 27 (right). (H) Bioluminescence imaging of U87 shVMP1 and vector orthotopic xenografts at Day 28. (I) Representative images of hematoxylin and eosin‐stained sections at Day 28 post‐injection. Scale bar, 2000 µm. (J) Kaplan–Meier survival analysis of mice with U87 shVMP1 and vector intracranial xenografts ( n = 5). (K) Representative western blot images of U87 shVMP1 and vector subcutaneous xenografts showing the expression of autophagy markers p62 and LC3 I/II (left). Quantification of band intensities normalized to GAPDH (right). ns, no statistical significance; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Human embryonic kidney cells 293T (293T) and human GBM cell lines U87 and U251 were purchased from the American Type Culture Collection (ATCC).

Techniques: Western Blot, Transmission Assay, Electron Microscopy, Expressing, Knockdown, Control, Plasmid Preparation, Injection, Imaging, Staining

Journal: MedComm

Article Title: Integrative Single‐Cell Analysis Reveals Targetable Vacuole Membrane Protein 1‐Mediated Mechanism of Tumor Angiogenesis in Glioblastoma

doi: 10.1002/mco2.70619

Figure Lengend Snippet: VMP1 mediates angiogenesis and vascular permeability through activation of endothelial cells in the TME. (A) Representative western blot image (top) and quantification (bottom) of VEGFR2 expression in human primary endothelial cells (HUVEC) after culturing with conditioned medium (CM) collected from VMP1‐overexpressing glioblastoma cell lines U87 and U251. (B) Representative immunofluorescence image of VEGFR2 expression (red) and DAPI (blue) in HUVEC cultured with conditioned medium. Scale bar, 200 µm. (C) Representative immunofluorescence staining image of VE‐cadherin expression (red) and DAPI (blue) in HUVEC with CM. Scale bar, 200 µm. (D) Human protein angiogenesis array showing 55 angiogenesis‐related proteins in the CM collected. (E) Quantification of eight of the angiogenesis‐related proteins, including tissue factor (TF), granulocyte‐macrophage colony stimulating factor (GM‐CSF), macrophage inflammatory protein 1α (MIP1α), Serpin E1, Thrombospondin‐1 (THBS1), Angiogenin, tissue inhibitor of metalloproteinase 1 (TIMP‐1), and VEGF‐C. (F) Spatial distribution of spot degree between VMP1 high cancer cells and endothelial cells in the spatial mRNA dataset. (G–I) Spatial distribution of angiogenesis (G), Serpin E1 (H), and TIMP1 (I) expression in the spatial mRNA dataset. * p < 0.05; ** p < 0.01; *** p <0 .001.

Article Snippet: Human embryonic kidney cells 293T (293T) and human GBM cell lines U87 and U251 were purchased from the American Type Culture Collection (ATCC).

Techniques: Permeability, Activation Assay, Western Blot, Expressing, Immunofluorescence, Cell Culture, Staining

Journal: MedComm

Article Title: Integrative Single‐Cell Analysis Reveals Targetable Vacuole Membrane Protein 1‐Mediated Mechanism of Tumor Angiogenesis in Glioblastoma

doi: 10.1002/mco2.70619

Figure Lengend Snippet: Targeted inhibition of VEGFA represses VMP1‐mediated tumor growth. (A) The treatment timeline and bioluminescence detection of mice treated with bevacizumab (BEV) at different time points (Day 3, Day 7, and Day 13). (B) Bioluminescence detection of U87 tumor‐bearing mice treated with BEV and vehicle. (C) Hematoxylin and eosin staining of mice brain, dotted area indicates the tumor region. Scale bar, 1000 µm. (D) Relative total photon flux in orthotopic mice model after treatment. (E) Changes in body weight in mice after treatments. (F) Kaplan–Meier survival of mice after being treated with bevacizumab and vector control. ns, no statistical significance.

Article Snippet: Human embryonic kidney cells 293T (293T) and human GBM cell lines U87 and U251 were purchased from the American Type Culture Collection (ATCC).

Techniques: Inhibition, Staining, Plasmid Preparation, Control

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation at S1288 of leukemia associated RhoGEF (LARG/ARHGEF12) induces plasma membrane localization and promotes binding and activation of RhoA

doi: 10.1016/j.jbc.2025.110996

Figure Lengend Snippet: EGFR activation leads to phosphorylation of LARG at S1288 through RSK . A , schematic representation of of the LARG protein showing the functional domains: PDZ, RH, DH, PH, and CC. These domains coordinate LARG activation, RhoA interaction and localization. B , posttranslational modifications determined by Mass Spec are shown as curated by PhosphoSitePlus ( www.phosphosite.com ). The height of the lines relates to the number of reports in which that modification was identified. C , Western blot analysis of LARG phosphorylation from U87 MG cells transfected with Myc-LARG-WT, Myc-LARG-S1288 A, or Myc-EV for 48h. Cells were starved for 24h, then treated with EGF (100 ng/ml) for 10 min. Cell lysates were immunoprecipitated with a Myc antibody, and LARG phosphorylation was detected using a phospho-AKT substrate antibody. LARG phosphorylation was quantified relative to Myc tag expression ( right ). D , starved cells were subjected to RSK inhibitors B-ID1780 or PMD-026 for 24h or 4h before treatment with 100 ng/ml EGF for 10 min. LARG, RSK and YB1 phosphorylation levels were quantified as fold changes ( right ). Results are representative of three independent experiments, each of which was done in triplicate and presented as the mean ± standard deviation. ∗ p ˂ 0.05.

Article Snippet: Human GBM U87 MG cells were obtained from the National Cancer Institute’s Tumor Repository and the cell line identity was validated by STR profiling by ATCC and tested negative for mycoplasma (LT07–188; Lonza).

Techniques: Activation Assay, Phospho-proteomics, Functional Assay, Mass Spectrometry, Modification, Western Blot, Transfection, Immunoprecipitation, Expressing, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation at S1288 of leukemia associated RhoGEF (LARG/ARHGEF12) induces plasma membrane localization and promotes binding and activation of RhoA

doi: 10.1016/j.jbc.2025.110996

Figure Lengend Snippet: Phosphorylation at S1288 is required for LARG to induce cell motility . A , U87 MG cells and B , GBM39 cells were transfected with LARG-WT, LARG-S1288 A, LARG-delC, or Myc-EV for 48h. Transfected cells were serum-starved before the assay. Cell migration was assessed by Transwell assay ( top ), and cell invasion was assessed by Transwell assay with matrigel ( bottom ). C , the LARG-specific inhibitor Y16 (10 μM) was used to test cell migration ( top ) and invasion ( bottom ) of U87 MG cells transfected with Myc-LARG-WT or Myc-EV. D , endogenous LARG activity was tested in U87 MG and GBM39 cells using a wound healing assay. The migration rate was calculated as Wound Healing (%) = (Initial wound area - Final wound area)/Initial wound area × 100. Scale bar 100 μm. Results are representative of three independent experiments, each was done in triplicate and presented as the mean ± standard deviation. ∗ p ˂ 0.05.

Article Snippet: Human GBM U87 MG cells were obtained from the National Cancer Institute’s Tumor Repository and the cell line identity was validated by STR profiling by ATCC and tested negative for mycoplasma (LT07–188; Lonza).

Techniques: Phospho-proteomics, Transfection, Migration, Transwell Assay, Activity Assay, Wound Healing Assay, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation at S1288 of leukemia associated RhoGEF (LARG/ARHGEF12) induces plasma membrane localization and promotes binding and activation of RhoA

doi: 10.1016/j.jbc.2025.110996

Figure Lengend Snippet: EGF stimulation induces LARG membrane localization through phosphorylation at S1288 . A , U87MG cells were transfected with Myc-LARG-WT, Myc-LARG-S1288 A, or Myc-EV for 48h. Cells were starved for 24h before treatment with 100 ng/ml EGF for 10 min. Nuclear and cytosolic lysates were analyzed to determine LARG localization by immunoblotting with a Myc antibody. Histone H3 and α-tubulin antibodies were used as a control for nuclear and cytosol fractionation respectively. B–D , U87 MG cells were transfected with Myc-LARG-WT, Myc-LARG-S1288 A, Myc-LARG-delC, or Myc-EV for 48h. Cells were starved for 24h before treatment with 100 ng/ml EGF for 0- 5-10- and 120-min. Cells were stained with Myc antibody (red ) and DAPI ( blue ) respectively. B , confocal microscope was used to determine the localization of different LARG constructs. Arrows indicate nucleus. C , total Internal Reflection Fluorescence (TIRF) microscopy was used to visualize Myc-LARG membrane localization. Arrows indicate membrane localization. Scale bar 20 μm. All images are representative of at least three independent experiments. D , quantification of LARG fluorescence intensity obtained from TIRF microscopy. Each dot represents to an individual cell; ∗∗ p < 0.01. E , Western blot analysis was performed to validate the phosphorylation levels of Myc-LARG-WT and Myc-LARG-S1288 A following EGF stimulation at different incubation times, using a phospho-AKT substrate antibody.

Article Snippet: Human GBM U87 MG cells were obtained from the National Cancer Institute’s Tumor Repository and the cell line identity was validated by STR profiling by ATCC and tested negative for mycoplasma (LT07–188; Lonza).

Techniques: Membrane, Phospho-proteomics, Transfection, Western Blot, Control, Fractionation, Staining, Microscopy, Construct, Fluorescence, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation at S1288 of leukemia associated RhoGEF (LARG/ARHGEF12) induces plasma membrane localization and promotes binding and activation of RhoA

doi: 10.1016/j.jbc.2025.110996

Figure Lengend Snippet: LARG phosphorylation at S1288 induces its ability to bind and activate RhoA . A , U87MG cells were transfected with Myc-LARG-WT, Myc-LARG-S1288 A, or Myc-EV for 48h. Protein - protein interaction of endogenous RhoA with exogenous LARG-WT or S1288 A mutant was determined by proximity ligation assay. Nuclei were counterstained with DAPI ( blue ). Red fluorescent dots represent PLA signals indicating the interaction between RhoA and Myc-LARG-WT or S1288 A. Expression of Myc-LARG-WT and S1288 A in U87 cells was confirmed by immunoblotting. All images are representative of four independent experiments. Scale bar 20 μm ∗∗ p < 0.05. B , U87 MG cells were co-transfected with Myc-LARG-WT, LARG-S1288 A, or Myc-EV along with HA-RhoA-WT or HA-EV for 48h and starved for 24h, then treated with or without 100 ng/ml EGF for 10 min. The interaction between LARG and RhoA was determined by immunoprecipitating Myc tagged LARG and immunoblotting for HA-RhoA. n = 3. C , U87 MG cells were transfected with Myc-LARG-WT, LARG-S1288 A, Myc-LARG-delC, or Myc-EV for 48h and starved for 24h, then treated with 100 ng/ml EGF for 10 min vs nontreated cells. The interaction was assessed by incubating total cell lysates with beads bound to GST-RhoA or GST-vector, followed by immunoblotting for Myc antibody. Results are representative of three independent experiments. D , U87 MG cells were transfected with Myc-LARG-WT or Myc-LARG-S1288A for 48h. Cells were then serum-starved for 24h and treated with EGF for 120 min. Cells were then fixed and stained with anti-Myc ( green ) and anti-RhoA-GTP ( red ) antibodies. DAPI was used as a nuclear counterstain ( blue ). Images are representative of two independent experiments. White boxes highlight the RhoA-GTP signal in the LARG-transfected cells. Scale bar 50 μm. E , Nikon NIS-Elements software was used to trace the perimeter of all LARG-transfected cells and to quantify the mean cellular fluorescent intensity of the RhoA-GTP signal. Mean cellular fluorescent intensity was graphed for all WT-LARG untreated cells (n = 7), WT-LARG EGF-treated cells (n = 28), LARG-S1288 A untreated cells (n = 7), and LARG-S1288 A EGF-treated cells (n = 31). Statistical significance was assessed via two-tailed t test (∗ p < 0.05).

Article Snippet: Human GBM U87 MG cells were obtained from the National Cancer Institute’s Tumor Repository and the cell line identity was validated by STR profiling by ATCC and tested negative for mycoplasma (LT07–188; Lonza).

Techniques: Phospho-proteomics, Transfection, Mutagenesis, Proximity Ligation Assay, Expressing, Western Blot, Plasmid Preparation, Staining, Software, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation at S1288 of leukemia associated RhoGEF (LARG/ARHGEF12) induces plasma membrane localization and promotes binding and activation of RhoA

doi: 10.1016/j.jbc.2025.110996

Figure Lengend Snippet: LARG Phosphorylation at S1288 does not affect its homo-oligomerization . U87 MG cells were transfected with Myc-LARG-WT, Myc-LARG-S1288 A, or Myc-EV for 24 h, followed by transfection with GFP-LARG-WT. The cells were incubated for an additional 48 h to achieve maximum protein expression. Complex formation was assessed by immunoprecipitating Myc-tagged LARG and performing immunoblotting for GFP. The phosphorylation levels of Myc-LARG-WT and Myc-LARG-S1288 A were confirmed by phospho-AKT substrate antibody. The results are representative of three independent experiments.

Article Snippet: Human GBM U87 MG cells were obtained from the National Cancer Institute’s Tumor Repository and the cell line identity was validated by STR profiling by ATCC and tested negative for mycoplasma (LT07–188; Lonza).

Techniques: Phospho-proteomics, Transfection, Incubation, Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation at S1288 of leukemia associated RhoGEF (LARG/ARHGEF12) induces plasma membrane localization and promotes binding and activation of RhoA

doi: 10.1016/j.jbc.2025.110996

Figure Lengend Snippet: CAAX-mediated membrane localization of LARG-S1288A increases co-localization with RhoA without affecting RhoA activation . U87MG cells were transfected with either LARG-S1288A or LARG-S1288A-CAAX to determine whether induced membrane localization of mutant LARG would activate RhoA. A , Addition of the CAAX motif to LARG-S1288 A increased membrane localization of protein, particularly in response to EGF stimulation. Transfected cells were serum-starved and treated with EGF for 0, 10 or 120 min. Cells were fixed and stained with anti-Myc ( green ) antibodies. DAPI was used as a nuclear counterstain ( blue ). TIRF imaging was performed to measure membrane localization of Myc-tagged LARG. Images are representative of two independent experiments with either TIRF images alone or merged TIRF/Myc/DAPI images. TIRF signal intensity for each Myc-tagged cell measured was graphed, and statistical significance between LARG-S1288 A and LARG-S1288A-CAAX was assessed for each time-point via two-tailed t test (∗ p < 0.05). B , LARG-S1288A-CAAX demonstrates increased co-localization with RhoA compared to LARG-S1288A. Transfected U87 MG cells were serum-starved and treated with EGF for 0 or 10 min, at which point they were fixed and stained with anti-Myc and anti-RhoA antibodies. Proximity ligation assay was used to visualize interactions between RhoA and Myc-tagged LARG-S1288 A or LARG-S1288A-CAAX, with red fluorescent dots marking points of interaction. Images are representative of two independent experiments. Scale bar 50 μm. Fluorescent dots per cell were quantified and graphed. Statistical significance between LARG-S1288 A and LARG-S1288A-CAAX was assessed for each time-point via two-tailed t test (∗ p < 0.05). C , Cells transfected with LARG-S1288A-CAAX show no increase in active RhoA-GTP expression compared to LARG-S1288A. Transfected U87 MG cells were serum-starved, then either untreated or treated with EGF 120 min. Cells were then fixed and stained with anti-Myc ( green ) and anti-RhoA-GTP ( red ) antibodies. DAPI was used as a nuclear counterstain ( blue ). Images are representative of two independent experiments. Scale bar 50 μm. Nikon NIS-Elements software was used to trace the perimeter of all LARG-transfected cells and to quantify the mean cellular fluorescent intensity of the RhoA-GTP signal. Mean fluorescent intensity of RhoA-GTP was graphed for each Myc-positive cell imaged. Statistical significance between EGF-treated and untreated cells was assessed via two-tailed t test ( p < 0.05).

Article Snippet: Human GBM U87 MG cells were obtained from the National Cancer Institute’s Tumor Repository and the cell line identity was validated by STR profiling by ATCC and tested negative for mycoplasma (LT07–188; Lonza).

Techniques: Membrane, Activation Assay, Transfection, Mutagenesis, Staining, Imaging, Two Tailed Test, Proximity Ligation Assay, Expressing, Software

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation at S1288 of leukemia associated RhoGEF (LARG/ARHGEF12) induces plasma membrane localization and promotes binding and activation of RhoA

doi: 10.1016/j.jbc.2025.110996

Figure Lengend Snippet: Endogenous LARG phosphorylation at S1288 and RhoA activation in GBM tissues . A , the phospho-LARG S1288 specific antibody was tested in U87MG cells transfected with Myc-LARG-WT, Myc-LARG-S1288 A, or Myc-EV. After 24 h of starvation, cells were treated with EGF for 10 min in the presence or absence of BI-D1870. B , the phospho-LARG S1288 antibody demonstrates specificity to the phosphorylated form of the protein. U87MG cells were transfected with Myc-LARG-WT of Myc-EV. After 24 h of starvation, cells were treated with EGF for 10 min. Protein was harvested and lysates were treated with λ phosphatase prior to immunoblotting. C , RSK2 phosphorylates LARG at S1288 in PDX-derived GBM39 cells. Cells maintained in complete medium with 10% FBS were treated with BI-D1870 for 4 h. Subsequently cells were starved in 0.1% FBS medium for 24 h prior to 10 min EGF treatment alone or after prior 4 h BID treatment. Immunoblotting was used to determine total expression and phosphorylation of target proteins. The results are representative of three independent experiments. D , levels of activated RhoA-GTP positively correlate with levels of phosphorylated LARG S1288 in human GBM tumor lysates. Endogenous total LARG, pLARG-S1288 and RhoA expression in seven human GBM tumor lysates were analyzed by immunoblotting. Activated RhoA-GTP was isolated by immunoprecipitation and detected with a RhoA antibody. Signals for each target protein expression were normalized to corresponding sample β-tubulin expression. A linear regression analysis was performed to assess the relationship between Activated RhoA expression and pLARG S1288 expression in all seven tumors.

Article Snippet: Human GBM U87 MG cells were obtained from the National Cancer Institute’s Tumor Repository and the cell line identity was validated by STR profiling by ATCC and tested negative for mycoplasma (LT07–188; Lonza).

Techniques: Phospho-proteomics, Activation Assay, Transfection, Western Blot, Derivative Assay, Expressing, Isolation, Immunoprecipitation

Journal: Journal of Inflammation Research

Article Title: Synthetic 1,3,6-Tri-O-Galloyl-α-D-Glucose Mimics the Hippo Pathway Inhibitor VT107 in Suppressing Concanavalin A-Induced Inflammation in Human Glioblastoma Cells

doi: 10.2147/JIR.S565721

Figure Lengend Snippet: Hippo pathway inhibitors alter the Concanavalin A-induced phosphorylation of ERK and IκB in human U87 glioblastoma cells . Serum-starved human U87 glioblastoma cells were treated with 30 µg/mL Concanavalin A (ConA) for 5 min in the absence or presence of 3 µM IAG933 (IAG), GNE7883 (GNE), or VT107 (VT). ( A ) Protein cell lysates were harvested and processed for immunoblotting as described in the Methods section, to detect the phosphorylation status of ERK and of IκB. ( B ) Densitometric analysis was performed and phosphorylation status expressed as the extent of phospho-ERK/ERK or phosphor-IκB/IκB ratios over untreated control cells. Data points are the means ± SEM of 3 independent experiments.

Article Snippet: Human U87 glioblastoma (GBM)-derived cells were sourced from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Phospho-proteomics, Western Blot, Control

Journal: Journal of Inflammation Research

Article Title: Synthetic 1,3,6-Tri-O-Galloyl-α-D-Glucose Mimics the Hippo Pathway Inhibitor VT107 in Suppressing Concanavalin A-Induced Inflammation in Human Glioblastoma Cells

doi: 10.2147/JIR.S565721

Figure Lengend Snippet: Time-dependent inhibition of Concanavalin A-induced phosphorylation of ERK and IκB by αTGG in human U87 glioblastoma cells . Serum-starved human U87 glioblastoma cells were treated with 30 µg/mL Concanavalin A (ConA) for the indicated time in the absence or presence of 30 µM αTGG. ( A ) Protein cell lysates were harvested and processed for immunoblotting as described in the Methods section, to detect the phosphorylation status of ERK and of IκB. ( B ) Densitometric analysis was performed and phosphorylation status expressed as the maximal value of phospho-ERK/ERK or phosphor-IκB/IκB ratios for control (grey shades) vs αTGG (black shades). Western blot and densitometric analysis are representative of 3 independent experiments.

Article Snippet: Human U87 glioblastoma (GBM)-derived cells were sourced from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Inhibition, Phospho-proteomics, Western Blot, Control

Journal: Journal of Inflammation Research

Article Title: Synthetic 1,3,6-Tri-O-Galloyl-α-D-Glucose Mimics the Hippo Pathway Inhibitor VT107 in Suppressing Concanavalin A-Induced Inflammation in Human Glioblastoma Cells

doi: 10.2147/JIR.S565721

Figure Lengend Snippet: Dose-dependent inhibition of Concanavalin A-induced phosphorylation of ERK and IκB by αTGG in human U87 glioblastoma cells . Serum-starved human U87 glioblastoma cells were treated with the indicated concentrations of Concanavalin A (ConA) for 5 min in the absence or presence of 30 µM αTGG. ( A ) Protein cell lysates were harvested and processed for immunoblotting as described in the Methods section, to detect the phosphorylation status of ERK and of IκB. ( B ) A representative densitometric analysis was performed and phosphorylation status expressed as the maximal value of phospho-ERK/ERK or phosphor-IκB/IκB ratios for control (closed circles) vs αTGG (open circles). Western blot and densitometric analysis are representative of 3 independent experiments.

Article Snippet: Human U87 glioblastoma (GBM)-derived cells were sourced from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Inhibition, Phospho-proteomics, Western Blot, Control

Journal: Journal of Inflammation Research

Article Title: Synthetic 1,3,6-Tri-O-Galloyl-α-D-Glucose Mimics the Hippo Pathway Inhibitor VT107 in Suppressing Concanavalin A-Induced Inflammation in Human Glioblastoma Cells

doi: 10.2147/JIR.S565721

Figure Lengend Snippet: Dose-dependent inhibition of Concanavalin A-induced Snail, COX2, and activation of proMMP-2 by αTGG in human U87 glioblastoma cells . Serum-starved human U87 glioblastoma cells were treated with the indicated concentrations of αTGG in the absence or presence of 30 µg/mL Concanavalin A (ConA) for 24 hours. ( A ) Protein cell lysates were harvested and processed for immunoblotting as described in the Methods section, to detect the expression levels of Snail and COX2. GAPDH served as a loading control. ( B ) A gelatin zymography was performed using the conditioned media isolated from the respective conditions as described in the Methods section to assess the effect of αTGG on the extent of ConA-induced proMMP-2 activation. ( C ) U87 glioblastoma cells were treated with the indicated concentrations of αTGG for 24 hours. Cell viability was evaluated for each concentration using the Trypan Blue exclusion assay and a TC20 Automated Cell Counter (Biorad). Data are representative of two independent experiments.

Article Snippet: Human U87 glioblastoma (GBM)-derived cells were sourced from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Inhibition, Activation Assay, Western Blot, Expressing, Control, Zymography, Isolation, Concentration Assay, Trypan Blue Exclusion Assay

Journal: Journal of Inflammation Research

Article Title: Synthetic 1,3,6-Tri-O-Galloyl-α-D-Glucose Mimics the Hippo Pathway Inhibitor VT107 in Suppressing Concanavalin A-Induced Inflammation in Human Glioblastoma Cells

doi: 10.2147/JIR.S565721

Figure Lengend Snippet: Differential YAP/TEAD control of Concanavalin A-mediated regulation of Hippo pathway downstream effectors . Serum-starved human U87 glioblastoma cells were treated with the indicated concentrations of ConA for 24 hours, total RNA was extracted and RT-qPCR performed as described in the Methods section. ( A ) Gene expression of inflammatory ( COX2, IL6 ) and downstream Hippo pathway effectors ( AXL, CRGF, CYR61 , and NF2 ). ( B ) Transient gene silencing of YAP1 and TEAD was performed and extent and specificity of gene repression compared to PPIA . ( C ) Impact of YAP1 and TEAD silencing on ConA-mediated effects on downstream Hippo pathway effectors. Data points are the means ± SEM of triplicates from independent experiments.

Article Snippet: Human U87 glioblastoma (GBM)-derived cells were sourced from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Control, Quantitative RT-PCR, Gene Expression

Journal: Journal of Inflammation Research

Article Title: Synthetic 1,3,6-Tri-O-Galloyl-α-D-Glucose Mimics the Hippo Pathway Inhibitor VT107 in Suppressing Concanavalin A-Induced Inflammation in Human Glioblastoma Cells

doi: 10.2147/JIR.S565721

Figure Lengend Snippet: The Hippo pathway inhibitor VT107 alters dose-dependently the Concanavalin A-induced phosphorylation of ERK and IκB in human U87 glioblastoma cells . Serum-starved human U87 glioblastoma cells were treated with 30 µg/mL Concanavalin A (ConA) for 5 min in the absence or presence of the indicated concentrations of VT107 (VT). ( A ) Protein cell lysates were harvested and processed for immunoblotting as described in the Methods section, to detect the phosphorylation status of ERK and of IκB. ( B ) A representative densitometric analysis was performed and phosphorylation status expressed as the percent of phospho-ERK/ERK or phospho-IκB/IκB ratios of ConA-treated cells. Western blot and densitometric analysis are representative of 3 independent experiments. ( C ) Conditioned media was collected and gelatin zymography performed as described in the Methods section to monitor the impact of VT107 on proMMP-2 activation.

Article Snippet: Human U87 glioblastoma (GBM)-derived cells were sourced from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Phospho-proteomics, Western Blot, Zymography, Activation Assay

Journal: Journal of Inflammation Research

Article Title: Synthetic 1,3,6-Tri-O-Galloyl-α-D-Glucose Mimics the Hippo Pathway Inhibitor VT107 in Suppressing Concanavalin A-Induced Inflammation in Human Glioblastoma Cells

doi: 10.2147/JIR.S565721

Figure Lengend Snippet: The Hippo pathway inhibitor VT107 and aTGG share a common anti-inflammatory molecular signature in Concanavalin A-primed human U87 glioblastoma cells . ( A ) Serum-starved human U87 glioblastoma cells were treated for 24 hours with 30 µg/mL Concanavalin A (ConA), total RNA extracted and processed for a gene array screen of 141 inflammatory-associated genes as described in the Methods section by RT-qPCR. ( B ) Serum starved cells were similarly treated with ConA in the presence of 1 µM of either IAG933 (IAG), GNE7883 (GNE) or VT107 (VT) for 24 hours and extent of inhibition of the 10 most induced common genes assessed and correlated to that of 30 µM αTGG. ( C ) Protein-to-protein interaction network of the 10 commonly shared and downregulated genes between all the inhibitors tested in ConA-treated cells as determined with STRING.

Article Snippet: Human U87 glioblastoma (GBM)-derived cells were sourced from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Quantitative RT-PCR, Inhibition