osteosarcoma cell line u2os  (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: Bone Research

Article Title: ADAR2 induces the differentiation of osteosarcoma cells by editing activity on IGFBP7: new implications for therapy

doi: 10.1038/s41413-026-00516-6

Figure Lengend Snippet: Effects of ADAR2 overexpression in osteosarcoma cell lines. a Real-Time RT-PCR and b Western Blot analysis of ADAR2 expression in MSC, osteoblasts (OB) and osteosarcoma cell lines Saos-2 and 143B. In b upper panels : representative blots; lower panel : densitometric analysis. c FACS analysis of the proliferative rate evaluated by CMAC staining and d cell cycle analysis of Saos-2 ( left panel ) and 143B ( right panel ) cells transfected with ADAR2-pEGFP-C3 (pADAR2), ADAR2 E/A-pEGFP-C3 (pADAR2 E/A) or Empty-pEGFP-C3 (pEmpty) vectors. ADAR2 E/A vector was generated by a single mutation in the catalytic domain of ADAR2. e Migration ability of transfected Saos-2 ( left panel) and 143B ( right panel) cells. f Transwell invasion assay of transfected Saos-2 ( left panel ) and 143B cells ( right panel ). g Representative blot and h densitometric analysis of Runx2 and Osx in transfected Saos-2 ( left panel) and 143B cells ( right panel ). Results are expressed as mean ± sd and are reported as individual data points of independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; vs pEmpty transfected cells. # P < 0.05; ## P < 0.01 vs pADAR2 transfected cells

Article Snippet: Commercially available human OS cell lines Saos-2 (HTB-85) and 143B (CRL-8303) were purchased from American Type Culture Collection (ATCC, Washington, NW, USA).

Techniques: Over Expression, Quantitative RT-PCR, Western Blot, Expressing, Staining, Cell Cycle Assay, Transfection, Plasmid Preparation, Generated, Mutagenesis, Migration, Transwell Invasion Assay

Journal: Bone Research

Article Title: ADAR2 induces the differentiation of osteosarcoma cells by editing activity on IGFBP7: new implications for therapy

doi: 10.1038/s41413-026-00516-6

Figure Lengend Snippet: Terminal osteogenic differentiation and increased drugs susceptibility in pADAR2-transfected Saos-2 cells. a – c Mineralization assay of Saos-2 cells transfected with pADAR2, pADAR2 E/A or pEmpty vectors. a Upper panels : Alizarin Red staining; lower panels : Von Kossa staining. b Absorbance analysis of Alizarin Red staining. c Densitometric analysis of Von Kossa-stained area. d – h Real-Time RT-PCR expression analysis of d COL1A2 , e DMP1 , f MEPE , g PRKCA and h NANOG . In ( b – h ) results are expressed as mean ± sd and are reported as individual data points of independent experiments. i Cell viability analysis of transfected Saos-2 cells treated for 6 days with increasing concentrations of MTX (0, 1, 5, 10, 50 and 100 nmol/L, left panel ) and of MS275 (0, 0.5, 1, 2.5, 5 and 10 μmol/L, right panel ) for 2 days. The concentration of drugs able to reduce by 50% (GI 50 ) cell viability is reported in the upper part of each graph. Results are expressed as mean ± sd of at least three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.000 1 vs pEmpty transfected cells. # P < 0.05; ## P < 0.01; ### P < 0.001; #### P < 0.000 1 vs pADAR2 transfected cells. j FACS analysis of apoptosis of transfected Saos-2 cells treated with the GI 50 calculated for pEmpty transfected Saos-2, or with Vehicle. Results are expressed as mean ± sd and are reported as individual data points of independent experiments. ** P < 0.01 vs Vehicle treated cells

Article Snippet: Commercially available human OS cell lines Saos-2 (HTB-85) and 143B (CRL-8303) were purchased from American Type Culture Collection (ATCC, Washington, NW, USA).

Techniques: Transfection, Mineralization Assay, Staining, Quantitative RT-PCR, Expressing, Concentration Assay

Journal: Bone Research

Article Title: ADAR2 induces the differentiation of osteosarcoma cells by editing activity on IGFBP7: new implications for therapy

doi: 10.1038/s41413-026-00516-6

Figure Lengend Snippet: In vivo experiments. Seven-weeks-old NSG male mice were intratibially injected with Saos-2 cells transfected with pADAR2, pADAR2 E/A or pEmpty vectors; after 12 weeks animals were sacrificed. a Representative X-Ray pictures of primary bone tumors. b Quantification of the tumor volume. c Number of animals with metastases in liver, lungs and kidneys at sacrifice. d Hematoxylin/Eosin staining of liver, lungs and kidney metastases. Nodules were indicated by black arrowheads. Number of metastases in e liver, f lungs and g kidneys. h Representative pictures of immunohistochemistry and i quantification of Ki67 in liver, lungs and kidneys metastases. Results are expressed as mean ± sd. * P < 0.05; ** P < 0.01 vs pEmpty cells injected mice. ## P < 0.01 vs pADAR2 cells injected animals

Article Snippet: Commercially available human OS cell lines Saos-2 (HTB-85) and 143B (CRL-8303) were purchased from American Type Culture Collection (ATCC, Washington, NW, USA).

Techniques: In Vivo, Injection, Transfection, Staining, Immunohistochemistry

Journal: Bone Research

Article Title: ADAR2 induces the differentiation of osteosarcoma cells by editing activity on IGFBP7: new implications for therapy

doi: 10.1038/s41413-026-00516-6

Figure Lengend Snippet: RNA-seq analysis. a Gene expression based heatmap showing the unique clusterization of ADAR2 transfected Saos-2 cells. Real-Time RT-PCR expression analysis of b COL4A1 , c SERPINH1 , d SWAP-70 and e TENM1 for transcriptional validation. f – h Editing analysis. Upper panels : Sequence chromatograms of the transcripts and editing levels of COPA , IGFBP7 and COG3 . Arrows indicate editing positions. Lower panels : percentage of editing in f COPA , g IGFBP7 and h COG3 transcripts. Results are expressed as mean ± sd and are reported as individual data points of independent experiments. * P < 0.05; ** P < 0.01; **** P < 0.000 1 vs pEmpty transfected cells. # P < 0.05; ## P < 0.01; ### P < 0.001 vs pADAR2 transfected cells

Article Snippet: Commercially available human OS cell lines Saos-2 (HTB-85) and 143B (CRL-8303) were purchased from American Type Culture Collection (ATCC, Washington, NW, USA).

Techniques: RNA Sequencing, Gene Expression, Transfection, Quantitative RT-PCR, Expressing, Biomarker Discovery, Sequencing

Journal: Bone Research

Article Title: ADAR2 induces the differentiation of osteosarcoma cells by editing activity on IGFBP7: new implications for therapy

doi: 10.1038/s41413-026-00516-6

Figure Lengend Snippet: IGF1R pathway analysis. a – f Investigation of IGF1R pathway in Saos-2 cells transfected with pADAR2, pADAR2 E/A or pEmpty vectors. a Representative plots and b – f densitometric analysis of b p-Igf1r, c p-Irs, d p-Akt (T308), e p-Akt (S473) and f p-p70. Results are expressed as mean ± sd and are reported as individual data points of independent experiments. ** P < 0.01; *** P < 0.00 1 vs pEmpty transfected cells. # P < 0.05; ## P < 0.01 vs pADAR2 transfected cells. g – o Effects of the treatment with WT- or K95R-IGFBP7 on Saos-2 cell line. g – l Analysis of IGF1R pathway in Saos-2 cells treated with 2 μg/ml of WT- or K95R-IGFBP7 compared to vehicle (Veh) treated cells. g Representative plots and h – l densitometric analysis of h p-Igf1r, i p-Irs, j p-Akt (T308), k p-Akt (S473) and l p-p70. m , n Western blot analysis of Runx2 in Saos-2 cells treated with WT- or K95R-IGFBP7 compared to vehicle (Veh) treated cells. m Representative blot and n densitometric analysis. o Proliferation rate of Saos-2 treated with WT- or K95R-IGFBP7 compared to vehicle (Veh) treated cells. Results are expressed as mean ± sd and are reported as individual data points of independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001 vs Vehicle treated Saos-2 cells. # P < 0.05; ### P < 0.001 vs WT-IGFBP7 treated cells

Article Snippet: Commercially available human OS cell lines Saos-2 (HTB-85) and 143B (CRL-8303) were purchased from American Type Culture Collection (ATCC, Washington, NW, USA).

Techniques: Transfection, Western Blot

Journal: Nature Communications

Article Title: Versatile and sensitive detection of mono- and poly(ADP-ribosyl)ation reveals XRCC1-dependent remodelling of PARP1 signalling

doi: 10.1038/s41467-026-71311-4

Figure Lengend Snippet: A Immunoblot analysis of in vitro ADP-ribosylated PARP1 with (mono-ADPr) or without (poly-ADPr) PARG using Poly-/ Pan-/ Mono-ADPr antibodies. Detection was done in parallel resulting in comparable signal intensities. Red colour represents saturated signal. N = 3. B Dot-blot analysis of in vitro auto-poly-ADPr of PARP1 in 1:2 dilution series using Poly-/ Pan-ADPr antibodies. Detection was done in parallel resulting in comparable signal intensities. Red colour represents saturated signal. N = 3. C Immunoblot analysis of in vitro ADP-ribosylated PARP1 with (Ser-ADPr) or without (Asp/Glut-ADPr) HPF1 using Poly-ADPr antibodies. Detection was done in parallel resulting in comparable signal intensities. N = 3. D Immunoblot analysis of SDS cell extracts from 2 mM H2O2-treated wild-type (WT) U2OS cells with and without 1 μM Olaparib treatment with the indicated antibodies. Detection was done in parallel resulting in comparable signal intensities. Red colour represents saturated signal. N = 3. E Immunoblot analysis of SDS cell extracts from untreated wild-type (WT) hTERT RPE1 cells with and without 1 μM Olaparib treatment for 1 h with the indicated antibodies. Detection was done in parallel, resulting in comparable signal intensities. N = 3. F Immunofluorescent staining of mono-/ poly-/ pan-ADPr in 2 mM H2O2-treated WT U2OS cells with and without 1 μM Olaparib treatment using the indicated antibodies. Detection was done on individual basis, signal intensities between different antibodies are not comparable. Signals are normalised to WT untreated conditions. Error bars represent SEM. Combined analysis of 3 Biological Replicates. Representative Images from 1 Biological Replicate. Scale bar, 10 μM. Source data are provided as a Source Data file.

Article Snippet: U2OS cell lines were obtained, authenticated by STR profiling and confirmed mycoplasma-free by ATCC cell line authentication service.

Techniques: Western Blot, In Vitro, Dot Blot, Staining

Journal: Nature Communications

Article Title: Versatile and sensitive detection of mono- and poly(ADP-ribosyl)ation reveals XRCC1-dependent remodelling of PARP1 signalling

doi: 10.1038/s41467-026-71311-4

Figure Lengend Snippet: A Schematic illustration of the affinity maturation process. Illustrations generated in Adobe Illustrator. Schematics adapted from Dauben et al. . B ELISA analysis of antibody specificities using the indicated antibodies (2 μg/mL) and biotinylated peptides (61 nM). Bars represent the arithmetic mean of 3 independent experiments. Error bars represent SD. C Left: Dot Blot analysis of isolated RNA from U2OS cells before and after glucose starvation. Right: Enzymatic digestion control of isolated RNA. Detection was done in parallel, resulting in comparable signal intensities. N = 3. D Immunoblot analysis of SDS cell extracts from 2 mM H2O2-treated wild-type (WT) U2OS cells with and without 1 μM Olaparib treatment with the indicated antibodies. Detection was done in parallel, resulting in comparable signal intensities. Red colour represents a saturated signal. N = 3. E Immunofluorescent staining of mono-ADPr in 2 mM H2O2-treated WT U2OS cells with and without 1 uM Olaparib treatment using the indicated antibodies. Detection was done in parallel, resulting in comparable signal intensities. Signals are normalised to WT untreated conditions. Error bars represent SEM. Combined Analysis of 3 Biological Replicates. Representative Images from 1 Biological Replicate. Scale bar, 10 μM. F Immunoblot analysis of H2SO4 cell extracts from 2 mM H2O2-treated wild-type (WT) U2OS cells with and without 1 μM Olaparib treatment with H3S10/S28-ADPr site-specific antibodies. Detection was done in parallel, resulting in comparable signal intensities. N = 3. G Immunoblot analysis of SDS cell extracts from 2 mM H2O2-treated wild-type (WT) U2OS cells with and without 1 μM PARG inhibition treatment with the indicated antibodies. Detection was done in parallel, resulting in comparable signal intensities. Red colour represents a saturated signal. N = 3. Source data are provided as a Source Data file.

Article Snippet: U2OS cell lines were obtained, authenticated by STR profiling and confirmed mycoplasma-free by ATCC cell line authentication service.

Techniques: Generated, Enzyme-linked Immunosorbent Assay, Dot Blot, Isolation, Control, RNA Detection, Western Blot, Staining, Inhibition