cell lines  (ATCC)

Journal: Clinical Cancer Research

Article Title: A Novel Potent and Selective GCN2 Inhibitor, APL-4098, Has Antileukemic Activity through Dysregulation of Mitochondrial Function

doi: 10.1158/1078-0432.CCR-25-1444

Figure Lengend Snippet: APL-4098 is a potent inhibitor of GCN2. A, APL-4098 molecular structure. B, Crystal structure of APL-4098 bound to human GCN2. Backbone of the GCN2 catalytic site (blue ribbon) and binding of APL-4098, with the extensive interaction network in the ATP site shown with dashed red lines (hydrogen bonds) and yellow lines (hydrophobic contacts). C, Biochemical, ADME, and PK profile of APL-4098. *All data generated from a rat intravenous/oral study: 1 mg/kg i.v. and 3 mg/kg orally. 1 Data generated from the i.v. study and 2 data generated from the oral study. D, APL-4098 inhibition of GCN2 kinase activity, measured as the level of eIF2α phosphorylation, using LanthaScreen FRET-based assay. Plot shows mean ± SEM from three representative experiments conducted in duplicate. E, U2OS cells were treated with APL-4098 in the presence or absence of borrelidin as indicated for 4 hours. Cell lysates were analyzed for phosphorylation of eIF2α using HTRF assay. Plot shows mean ± SEM from four individual experiments conducted in duplicate. F, U2OS cells were grown in glutamine-depleted medium and treated with APL-4098 as indicated for 4 hours. Cell lysates were analyzed by Western blotting.

Article Snippet: The osteosarcoma cell line U2OS (HTB-96, RRID: CVCL_0042) was obtained from ATCC and cultured in DMEM high glucose (Gibco, #41965-039) with 10% FBS (Gibco, #10270-106), 1 mmol/L sodium pyruvate (Gibco, #11360-039), 1% nonessential amino acids (Gibco, #11140-035), and antibiotics (penicillin/streptomycin 100 U/mL and 100 μg/mL; Gibco, #15140-122).

Techniques: Binding Assay, Generated, Inhibition, Activity Assay, Phospho-proteomics, HTRF Assay, Western Blot

Journal: Clinical Cancer Research

Article Title: A Novel Potent and Selective GCN2 Inhibitor, APL-4098, Has Antileukemic Activity through Dysregulation of Mitochondrial Function

doi: 10.1158/1078-0432.CCR-25-1444

Figure Lengend Snippet: APL-4098 is a selective inhibitor of GCN2. A, Eurofins KINOMEScan selectivity panel assay Treespot results for APL-4098 tested at 1 μmol/L. B, Eurofins biochemical K d concentration–response assay. C, U2OS cells were treated with APL-4098 or positive control dabrafenib (bioRxiv 2024.08.14.607626) in the presence of BtdCPU for 4 hours. Cell lysates were analyzed for phosphorylation of eIF2α using HTRF assay. Plot shows mean ± SEM from three individual experiments conducted in duplicate. D, U2OS cells were treated with APL-4098 or positive control GSK2606414 in the presence of thapsigargin for 4 hours. Cell lysates were analyzed for phosphorylation of eIF2α using HTRF assay. Plot shows mean ± SEM from three individual experiments.

Article Snippet: The osteosarcoma cell line U2OS (HTB-96, RRID: CVCL_0042) was obtained from ATCC and cultured in DMEM high glucose (Gibco, #41965-039) with 10% FBS (Gibco, #10270-106), 1 mmol/L sodium pyruvate (Gibco, #11360-039), 1% nonessential amino acids (Gibco, #11140-035), and antibiotics (penicillin/streptomycin 100 U/mL and 100 μg/mL; Gibco, #15140-122).

Techniques: Concentration Assay, Positive Control, Phospho-proteomics, HTRF Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Gene Expression Alterations Associated With Resveratrol‐Induced Antiproliferative Effects and S‐Phase Cell Cycle Arrest in Osteosarcoma Cancer Cells

doi: 10.1111/jcmm.71111

Figure Lengend Snippet: Hyperspectral Imaging (HSI) of RSV and intracellular localisation in both OS cell lines. (A) Dark‐field optical imaging captures the signal of pure RSV, and (B) the associated spectral library shows the intensity of scattered light across wavelengths. (C) Dark‐field images and Spectral Angle Mapping (SAM) analysis in OS cell lines (SAOS‐2 and U2‐OS) show no RSV‐associated signals in control samples (completely black images), while treated cells display coloured pixels matching the RSV spectral signature, confirming intracellular localisation of the compound. Imaging was performed using a 60× oil immersion objective to resolve RSV distribution and spectral characteristics. (D) Quantification of RSV‐positive pixels reveals a statistically significant increase in both treated OS cell lines, compared to controls (*** p < 0.001), confirming cellular uptake of RSV.

Article Snippet: In vitro assays were conducted using OS cell lines SAOS‐2 and U2‐OS (ATCC, Cat. no. HTB‐85; Cat. no. HTB‐96) [ ], along with human bone marrow‐derived mesenchymal stem cells (hBMSCs) (Lonza Milan, Italy, PT‐2501). hBMSCs were characterised by flow cytometry analysis (FCA) for MSC surface markers, including positive markers (CD29, CD73 and CD90) and negative markers (CD14 and CD45) [ ].

Techniques: Imaging, Optical Imaging, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Gene Expression Alterations Associated With Resveratrol‐Induced Antiproliferative Effects and S‐Phase Cell Cycle Arrest in Osteosarcoma Cancer Cells

doi: 10.1111/jcmm.71111

Figure Lengend Snippet: Effects of RSV on the proliferation, viability and cell cycle distribution of OS cell lines and hBMSCs. (A) The MTT assay was used to assess the effect of resveratrol (RSV, 1–1000 μM) on OS cell lines (SAOS‐2 and U2‐OS) and healthy hBMSCs over 24, 48 and 72 h. In SAOS‐2 cells, RSV reduced proliferation in a dose‐dependent manner, compared to control ( p < 0.001), with significant decreases at 100 and 1000 μM after 48 h, compared to other treatments (** p < 0.01). In U2‐OS cells, RSV significantly reduced viability at all concentrations, with more differences observed after 24 h at higher concentrations (*** p < 0.0001). hBMSCs showed no significant changes, except for a notable increase at 1000 μM (*** p < 0.0001). (B) The Live/Dead assay confirmed the cytotoxic effect of RSV (100 μM) on OS cells after 48 h, using green Cyto‐dye for live cells and red propidium iodide for dead cells. (C) Fluorescence image quantification showed a significant reduction in live cells (*** p < 0.0001) and an increase in dead cells (** p < 0.001; * p < 0.01) in RSV‐treated OS cell lines compared to the control. (D) The effect of RSV on the cell cycle was analysed by BrdU/PI staining and flow cytometry after 48 h of treatment with 100 μM. The cytogram displays that RSV treatment resulted in a significant accumulation of OS cells in the S phase and a decrease in the G0/G1 and G2/M phases, compared to untreated controls. (E) Statistical analysis confirmed a significant increase in the S phase (*** p < 0.0001) and a significant reduction in the G0/G1 phase (* p < 0.001; ***p < 0.0001) in both OS cell lines.

Article Snippet: In vitro assays were conducted using OS cell lines SAOS‐2 and U2‐OS (ATCC, Cat. no. HTB‐85; Cat. no. HTB‐96) [ ], along with human bone marrow‐derived mesenchymal stem cells (hBMSCs) (Lonza Milan, Italy, PT‐2501). hBMSCs were characterised by flow cytometry analysis (FCA) for MSC surface markers, including positive markers (CD29, CD73 and CD90) and negative markers (CD14 and CD45) [ ].

Techniques: MTT Assay, Control, Live Dead Assay, Fluorescence, Staining, Flow Cytometry

Journal: Journal of Cellular and Molecular Medicine

Article Title: Gene Expression Alterations Associated With Resveratrol‐Induced Antiproliferative Effects and S‐Phase Cell Cycle Arrest in Osteosarcoma Cancer Cells

doi: 10.1111/jcmm.71111

Figure Lengend Snippet: RSV induces apoptosis in OS cell lines. (A) Flow cytometry with Annexin V/PI staining was used to assess apoptosis in OS cells treated with 100 μM RSV for 48 h. The analysis identified early apoptotic (Annexin V+), late apoptotic (Annexin V+/PI+) and necrotic (PI+) cells, with Annexin V and PI intensities plotted on the X and Y axes, respectively. (B) Quantification showed that RSV significantly increased late apoptotic and necrotic cells in both OS cell lines (* p < 0.0001 for SAOS‐2 and ** p < 0.01 for U2‐OS), compared to control. A decrease in early apoptotic cells is observed in U2‐OS (** p < 0.01). (C) Gene expression profiling after RSV treatment revealed 18 differentially expressed apoptotic genes in SAOS‐2 (12 upregulated, 6 downregulated) and 21 in U2‐OS (10 upregulated, 11 downregulated), based on a Log 2 FC > 1 or < −1. (D) Gene Set Enrichment Analysis (GSEA) grouped these modulated genes into four categories: Positive regulators of apoptosis, negative regulators, caspases and death domain receptors, showing both up‐ and downregulated genes in each cell line. (E) A Venn diagram showed 11 apoptotic genes commonly modulated in both OS cell lines, with 7 unique to SAOS‐2 and 9 to U2‐OS, suggesting shared and cell‐specific mechanisms of RSV‐induced apoptosis. (F) Immunostaining for caspase‐3/7 revealed increased activation in both RSV‐treated OS cell lines after 48 h. (G) Fluorescence quantification using ImageJ confirmed a significant rise in activated caspase‐3/7 levels (** p < 0.001) in both RSV‐treated OS cells compared to controls.

Article Snippet: In vitro assays were conducted using OS cell lines SAOS‐2 and U2‐OS (ATCC, Cat. no. HTB‐85; Cat. no. HTB‐96) [ ], along with human bone marrow‐derived mesenchymal stem cells (hBMSCs) (Lonza Milan, Italy, PT‐2501). hBMSCs were characterised by flow cytometry analysis (FCA) for MSC surface markers, including positive markers (CD29, CD73 and CD90) and negative markers (CD14 and CD45) [ ].

Techniques: Flow Cytometry, Staining, Control, Gene Expression, Immunostaining, Activation Assay, Fluorescence

Journal: Journal of Cellular and Molecular Medicine

Article Title: Gene Expression Alterations Associated With Resveratrol‐Induced Antiproliferative Effects and S‐Phase Cell Cycle Arrest in Osteosarcoma Cancer Cells

doi: 10.1111/jcmm.71111

Figure Lengend Snippet: RSV inhibits cell migration and modulates ECM related gene expression in OS cells lines. (A) Bright‐field images from a wound healing assay show that control OS cells fully close the wound by 72 h (T3), whereas RSV‐treated cells (100 μM) exhibit no wound closure at any time point 0‐72 h (T0–T3), indicating that RSV strongly inhibits cell migration. (B) Quantitative analysis confirms significant wound closure in control cells over time compared to baseline T0 (0h) (° p < 0.0001), with additional increases at 48 h (T2) and 72 h (T3) compared to 24 h (T1) (* p < 0.001). Complete closure is observed at 72 h (T3) in control cells. (C) ECM‐related gene expression analysis using RT 2 Profiler PCR Array shows differential expression in RSV‐treated cells: 43 genes are modulated in SAOS‐2 (29 upregulated, 14 downregulated) and 26 in U2‐OS (11 upregulated, 15 downregulated). (D) Gene Set Enrichment Analysis (GSEA) categorizes these genes into five functional groups: Cell–cell adhesion, ECM‐cell adhesion, ECM constituents, ECM remodelling and basement membrane components. Up‐ and downregulated genes are identified for each group in both cell lines. (E) A Venn diagram reveals 20 ECM‐related genes commonly modulated in both SAOS‐2 and U2‐OS, indicating shared pathways influenced by RSV, particularly those involved in cell adhesion and ECM remodelling.

Article Snippet: In vitro assays were conducted using OS cell lines SAOS‐2 and U2‐OS (ATCC, Cat. no. HTB‐85; Cat. no. HTB‐96) [ ], along with human bone marrow‐derived mesenchymal stem cells (hBMSCs) (Lonza Milan, Italy, PT‐2501). hBMSCs were characterised by flow cytometry analysis (FCA) for MSC surface markers, including positive markers (CD29, CD73 and CD90) and negative markers (CD14 and CD45) [ ].

Techniques: Migration, Gene Expression, Wound Healing Assay, Control, Quantitative Proteomics, Functional Assay, Membrane

Journal: Journal of Cellular and Molecular Medicine

Article Title: Gene Expression Alterations Associated With Resveratrol‐Induced Antiproliferative Effects and S‐Phase Cell Cycle Arrest in Osteosarcoma Cancer Cells

doi: 10.1111/jcmm.71111

Figure Lengend Snippet: RSV modulates the Wnt/β‐Catenin signalling pathway and affects vimentin expression and β‐catenin localisation in OS cells lines. (A) Real‐time PCR analysis shows that RSV treatment (100 μM, 48 h) significantly downregulates key genes of the Wnt/β‐catenin pathway in SAOS‐2 and U2‐OS cells, including CTNNB1 , MMP7 , MMP9 and CD44 (* p < 0.001), all associated with ECM degradation, stemness and invasiveness. Conversely, CDH1 (epithelial marker) is upregulated in both lines (* p < 0.001 for SAOS‐2 and ** p < 0.01 for U2‐OS), suggesting a shift toward an epithelial phenotype. WNT1 and VIM are significantly downregulated (* p < 0.01 and * p < 0.05, respectively) in both cell lines, with c‐MYC reduced in SAOS‐2 (* p < 0.01). (B) Immunocytochemistry reveals a notable decrease in Vimentin protein levels in RSV‐treated cells. Vimentin, a mesenchymal marker, appears less expressed, with treated cells showing morphological changes including elongated filaments and enlarged cell body and nucleus. (C) β‐catenin immunostaining indicates that RSV prevents its nuclear translocation, with the protein mainly localized at cell junctions and in the cytoplasm in treated cells, while in controls β‐catenin is predominantly nuclear, confirming RSV‐mediated inhibition of Wnt/β‐catenin signalling.

Article Snippet: In vitro assays were conducted using OS cell lines SAOS‐2 and U2‐OS (ATCC, Cat. no. HTB‐85; Cat. no. HTB‐96) [ ], along with human bone marrow‐derived mesenchymal stem cells (hBMSCs) (Lonza Milan, Italy, PT‐2501). hBMSCs were characterised by flow cytometry analysis (FCA) for MSC surface markers, including positive markers (CD29, CD73 and CD90) and negative markers (CD14 and CD45) [ ].

Techniques: Expressing, Real-time Polymerase Chain Reaction, Marker, Immunocytochemistry, Immunostaining, Translocation Assay, Inhibition